WHO Technical Report Series 922

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, with a view to recommending acceptable daily intakes (ADIs) and to prepare specifications for the identity and purity of food additives. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of food additives (including flavouring agents) and contaminants, assessments of intake, and the establishment and revision of specifications for food additives. A summary follows of the Committee’s evaluations of toxicological and intake data on various specific food additives (a-amylase from Bacillus lichenformis containing a genetically engineered a-amylase gene from B. licheniformis, annatto extracts, curcumin, diacetyl and fatty acid esters of glycerol, D-tagatose, laccase from Myceliophthora thermophila expressed in Aspergillus oryzae, mixed xylanase, b-glucanase enzyme preparation produced by a strain of Humicola insolens, neotame, polyvinyl alcohol, quillaia extracts and xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum), flavouring agents, a nutritional source of iron (ferrous glycinate, processed with citric acid), a disinfectant for drinking-water (sodium dichloroisocyanurate) and contaminants (cadmium and methylmercury). Annexed to the report are tables summarizing the Committee’s recommendations for ADIs of the food additives, recommendations on the flavouring agents considered, and tolerable intakes of the contaminants considered, changes in the status of specifications and further information requested or desired.

EVALUATION OF CERTAIN FOOD ADDITIVES AND CONTAMINANTS

EVALUATION OF CERTAIN FOOD ADDITIVES AND CONTAMINANTS WHO Technical Report Series — 922

Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives

World Health Organization Geneva
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This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization or of the Food and Agriculture Organization of the United Nations

WHO Technical Report Series 922

EVALUATION OF CERTAIN FOOD ADDITIVES AND CONTAMINANTS

Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives

World Health Organization Geneva 2004
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The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country.int). Italy) Evaluation of certain food additives and contaminants : sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives. 922) 1.Food additives — toxicity 2. Switzerland (tel: +41 22 791 2476. or concerning the delimitation of its frontiers or boundaries.Food contamination 4. territory.int).Risk assessment I. This publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization.WHO Library Cataloguing-in-Publication Data Joint FAO/WHO Expert Committee on Food Additives (2003 : Rome.Food additives — analysis 3. email: permissions@who. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned.Title II. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. Publications of the World Health Organization can be obtained from Marketing and Dissemination. (WHO technical report series . Errors and omissions excepted. The World Health Organization does not warrant that the information contained in this publication is complete and correct and shall not be liable for any damages incurred as a result of its use. the names of proprietary products are distinguished by initial capital letters.Flavoring agents — analysis 5. 20 Avenue Appia. World Health Organization. at the above address (fax: +41 22 791 4806. Requests for permission to reproduce or translate WHO publications — whether for sale or for noncommercial distribution — should be addressed to Publications. Typeset in China Printed in Switzerland ii . city or area or of its authorities. 1211 Geneva 27. email: bookorders@who. fax: +41 22 791 4857.Series ISBN 92 4 120922 4 ISSN 0512-3054 (NLM Classification: WA 712) © World Health Organization 2004 All rights reserved.

1.3 Joint FAO/WHO Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food 2.1.5.9 Polyvinyl alcohol 3. b-glucanase enzyme preparation.6 Talc 3. 9 11 18 22 23 26 28 30 35 37 40 42 42 42 43 44 44 44 45 iii .2.1.6.1.3 Specifications of purity for flavouring agents 2.2 Revision of specifications 3.11 Xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum 3.5 Food additive specifications 2.1.2.1 Use of proposed maximum limits in the intake assessment of food additives 2.2.2 Principles governing the toxicological evaluation of compounds on the agenda 2.5.2.6 Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae 3.4 Diacetyltartaric and fatty acid esters of glycerol 3.4 Natamycin 3. Introduction General considerations 2.6 Intake assessment of food additives 2.5 D-Tagatose 3.5. 3.2 Annatto extracts 3.1 b-Carotene from Blakeslea trispora 3.2. licheniformis 3.1.2 Residual solvents 2. produced by a strain of Humicola insolens 3.2 Consideration of the Guidelines Specific food additives (other than flavouring agents) 3.1 Compendium of Food Additive Specifications and Guide to Specifications 2.2.8 Neotame 3.3 Curcumin 3.1 Modification of the agenda 2.2 Safety evaluation of flavouring agents 2.7 Mixed xylanase.1 Chemical and technical assessments of food additives 2.2 Magnesium silicate (synthetic) 3.3 Revision of limits for metals in food additives 1 1 2 2 2 3 5 6 6 6 7 7 8 8 8 9 9 2.1.2.1 a-Amylase from Bacillus licheniformis containing a genetically engineered a-amylase gene from B.5 Sucrose esters of fatty acids 3.10 Quillaia extracts 3.2.1.1.1.6.Contents 1.4 Provision of scientific advice by FAO and WHO 2.1 Safety evaluations 3.3 Monomagnesium phosphate and trisodium diphosphate 3.1.

4 Estimated dietary intake 7.6 Linear and branched-chain aliphatic. unconjugated alcohols.1.2.1.2 Revision of existing specifications for flavouring agents Nutritional source of iron 5.2.1.1. acids and related esters: additional compounds 4.1 Cadmium 7. 7. acids and esters 4.4. di.1.4 Dose-response assessments 7.1 Introduction 7. a. 9.1.2 Aliphatic. alicyclic-fused and aromatic-fused ring lactones 4.1 Specifications for flavouring agents evaluated for the first time at the sixty-first meeting 4.2.3 Observations in humans 7.5 Hydroxypropenylbenzenes 4.2 Observations in animals 7.1.1 Sodium dichloroisocyanurate Contaminants 7.2 Observations in animals 7.1 Ferrous glycinate (processed with citric acid) Disinfectant for drinking-water 6.b-unsaturated.3 Aliphatic branched-chain saturated and unsaturated alcohols.7 Simple aliphatic and aromatic sulfides and thiols: additional compounds 4. Flavouring agents 4. Acknowledgements References Annex 1 Reports and other documents resulting from previous meetings of the Joint FAO/WHO Expert Committee on Food Additives iv 144 . alicyclic.1 Introduction 7.2. 6.1.1.2.2 Methylmercury 7.5 Estimated dietary intake 7.1.2. aldehydes.2.2 Revision of certain specifications for purity of flavouring agents 4.1. aldehydes.2.6 Evaluation Future work Recommendations 45 45 49 64 75 86 96 101 111 121 121 121 121 121 123 123 127 127 127 127 129 130 131 132 132 132 133 135 138 138 140 140 141 141 5.3 Observations in humans 7.1. and related esters 4.4 Aliphatic and aromatic ethers 4.1 Flavouring agents evaluated by the Procedure for the Safety Evaluation of Flavouring Agents 4. 8. acids. linear. unsaturated.1 Alicyclic.and trienals and related alcohols.5 Evaluation 7.

other toxicological information and information on specifications Annex 3 Further information required or desired Annex 4 Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less 153 163 164 v .Annex 2 Acceptable daily intakes.

C. Emeritus Professor of Food Science. Canberra. Massachusetts. Al Ain. NSW.J.M. London. National Institute of Public Health and Environmental Protection (RIVM). Woonona. Belgium Professor R. Bilthoven. SAFE Consortium on Food Safety.A. 10–19 June 2003 Members Dr C. England Dr D. AOCS. Danish Veterinary and Food Administration. Harvard Medical School. Food Control.J. National Food Agency. Center for Food Safety and Nutrition. USA (WHO Temporary Adviser) Dr D. Children’s Hospital. Maryland.G. National Institute for Agricultural Research. Champaign Illinois. USA Mrs I. UK Food Standards Agency. Vavasour. Japan Dr P. Centre for Substances and Integrated Risk Assessment. Swiss Federal Office of Public Health. Pascal. Agriculture and Fisheries. USA Dr Y. Finland Dr D. Kuznesof. Cheshire. Maryland. Tokyo. Office of Food Additive Safety.Benford. Whitehouse. Hattan. Center for Food Safety and Applied Nutrition. University of Surrey. Switzerland Dr G. Brussels. Institut National de la Recherche Agronomique (INRA). Food Directorate. Guildford. College Park. Helsinki. Wallin. Zürich.B. Verger. Food Standards Australia New Zealand (FSANZ). Health Canada. Food and Drug Administration. Meyland. England (Vice-Chairman) Dr H. Australia (WHO Temporary Adviser) Dr D. Boston. School of Biomedical and Life Sciences. Canada (Rapporteur) Dr P. Fisher. Bellinger. Ministry of Food. Paris. England Secretariat Dr P. United Arab Emirates University. Bowdon. Food Toxicology Section. Speijers. Brooke-Taylor. Kawamura. Australia (WHO Temporary Adviser) Dr R. Office of Food Additive Safety. College Park. Veerabhadra Rao. United Arab Emirates Dr J. Surrey.E. Ontario.Sixty-first meeting of the Joint FAO/WHO Expert Committe on Food Addtives Rome. England (WHO Temporary Adviser) Dr S. Denmark (Chairman) Dr G. Walker. ACT. France Dr M. Schlatter. National Institute of Health Sciences. Central Laboratories Unit. Cantrill. Hatfield. Ottawa. Søborg. Food and Drug Administration. Hertfordshire.C. Abbott. Netherlands Mrs E. USA (FAO Consultant) vi .

Division of Risk Assessment. Viñuela. Division of Environmental Immunology and Toxicology. Jichi Medical School. Kroes. Center for Substances and Integrated Risk Assessment. Olempska-Beer. Food and Drug Administration. Lawrie. National Institute for Public Health and the Environment. Maryland.Dr M. Switzerland (WHO Staff Member) Mr J. France (Editor) Dr G. Netherlands (WHO Temporary Adviser) vii . Luetzow. International Programme on Chemical Safety. Buckinghamshire. Feeley.M.E.L. Mattia. England (WHO Temporary Adviser) Mr M. Institute for Risk Assessment Sciences. DiNovi. College Park. Moreau. WHO. Santiago. International Programme on Chemical Safety. Geneva. Japan (WHO Temporary Adviser) Dr Z. Page. Ehara. USA (FAO Consultant) Dr S. WHO. Mattock. UK Food Standards Agency. Food Directorate.C. Toxicological Evaluation Section. Canada (WHO Temporary Adviser) Dr A. Health Canada. FAO. Division of Biotechnology and GRAS Notice Review. Rome. Utrecht University. Maryland. Office of Food Additive Safety. USA (WHO Temporary Adviser) Dr H. Canada (FAO Consultant) Dr G. Geneva. Novel Foods Division. Food Quality and Standards Service. Bureau of Chemical Safety. Nishikawa. Leclercq. Munro. St Jean d’Ardières. Ottawa. CanTox Health Sciences International. Ontario. Italy (FAO Consultant) Dr E.K. Food and Drug Administration. Chile (FAO Consultant) Dr M. England (FAO Consultant) Dr C. Health Canada. Center for Food Safety and Applied Nutrition. Egan. Office of Food Additive Safety. Tokyo. Moy. Food and Drug Administration. Switzerland (Acting Joint Secretary) Mrs I. Netherlands (WHO Temporary Adviser) Dr C. Maryland. Center for Food Safety and Applied Nutrition. National Public Health Institute. Ontario. USA (WHO Temporary Adviser) Mr T. Pronk. Kayama. Canada (WHO Temporary Adviser) Professor F. Rome. Center for Food Safety and Applied Nutrition. Switzerland (WHO Staff Member) Dr I. Food and Nutrition Division. Soest. College Park. Fawell. Mississauga. College Park.J. WHO. Geneva. Ontario. Bilthoven. Italy (Joint Secretary) Dr A.A. Maryland. Division of Pathology. Center for Food Safety and Applied Nutrition. Office of Food Additive Safety. Japan (WHO Temporary Adviser) Professor R. USA (WHO Temporary Adviser) Ms S. National Research Institute for Food and Nutrition (INRAN). Bureau of Chemical Safety. College Park. Ottawa. Food and Drug Administration. Food Safety Department. Tochigi. Department of Health Science. National Institute of Health Sciences. London. Food Directorate.

Netherlands (WHO Temporary Adviser) Professor I. Environmental Health Sciences Division. Resnik. Tucson. Prince Edward Island Food Technology Centre. Pcia de Buenos Aires.K.G. Tritscher. National Institute for Environmental Studies. Charlottetown. Williams. Tohyama.G. New York. Technical Secretariat CCFAC. Clinical Pharmacology Group. Tsukuba. USA (WHO Temporary Adviser) * Appointed WHO Joint Secretary viii . Scheidegger. Canada (FAO Consultant) Professor I. La Plata. PE. Southampton. Société Générale de Surveillance. Institute of Bromatology. Department of Environmental Pathology and Toxicology. Serbia and Montenegro (FAO Consultant) Dr C. Switzerland (WHO Temporary Adviser)* Professor G. Ministry of Agriculture. Stankovic. The Hague.L. Nestlé (NRC-QS) Lausanne. England (WHO Temporary Adviser) Dr S. Department of Quality and Safety Assurance. Gurgaon (Haryana). University of Arizona. Japan (WHO Temporary Adviser) Dr A. USA (WHO Temporary Adviser) Dr J. Renwick. Argentina (FAO Consultant) Dr S. Belgrade. New York Medical College. Faculty of Pharmacy. Department of Pharmacology. Smith. Comision de Investigaciones Cientificas. Saxena. Valhalla. Arizona. College of Medicine. University of Southampton. Nature Management and Food Quality. India (FAO Consultant) Ms N.Professor A. Sipes.

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in press. Addendum 11. 52. 2003. No. One of the main objectives of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Specifications are issued separately by FAO under the title: Compendium of food additive specifications. No. x . FAO Food and Nutrition Paper. The IPCS is a joint venture of the United Nations Environment Programme. the International Labour Organization and the World Health Organization. Add. INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY The preparatory work for toxicological evaluations of food additives and contaminants by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) is actively supported by certain of the Member States that contribute to the work of the International Programme On Chemical Safety (IPCS).Monographs containing summaries of relevant data and toxicological evaluations are available from WHO under the title: Safety evaluation of certain food additives and contaminants. WHO Food Additive Series. 52. 11.

and — to undertake a toxicological evaluation of a disinfectant for drinking-water (section 6). a significant increase of staff and non-staff resources was being negociated for the forthcoming years. — to review and prepare specifications for selected food additives and flavouring agents (sections 3 and 4. de Haen. Assistant Director-General. FAO and WHO were committed to increasing efforts and resources to improve the provision of this advice. The meeting was opened by Mr H. on behalf of the DirectorsGeneral of the Food and Agriculture Organization of the United Nations and the World Health Organization. Mr de Haen made reference to the recently completed evaluation of the work of the Joint FAO/WHO Food Standards Programme (Codex Alimentarius Commission) and of this Committee and other joint FAO/WHO activities in providing scientific advice to Member countries. — to undertake toxicological evaluations of certain food additives. 1 . and Annex 2).1. General considerations As a result of the recommendations of the first Joint FAO/ WHO Conference on Food Additives. The tasks before the Committee were: — to elaborate further principles for evaluating the safety of food additives and contaminants (section 2). reference 160). held in September 1955 (1). 2. within FAO. and Annex 2). — to undertake a toxicological evaluation of a nutritional source of iron (section 5). FAO and WHO would report on steps underway to improve the work of the scientific expert committees and ad hoc consultations that provide scientific advice to Codex committees and to FAO/WHO Member countries. The present meeting was convened on the basis of the recommendation made at the fifty-ninth meeting (Annex 1. He noted that at the forthcoming twenty-sixth session of the Codex Alimentarius Commission. Introduction The Joint FAO/WHO Expert Committee on Food Additives met in Rome from 10 to 19 June 2003. there have been sixty previous meetings of the Expert Committee (Annex 1). FAO. flavouring agents and contaminants (sections 3. 4 and 7.

88. 70. 919 and 925 were removed from the agenda because the data necessary to establish full specifications were not available. 131. which should also form the basis for intake assessment.1 Chemical and technical assessments of food additives At previous meetings. No.2. 2. as well as the principles elaborated subsequently at a number of its meetings (Annex 1. 143. The document is prepared by an expert assigned before the meeting and is intended to provide to the Committee the basic information regarding the identity. 122. Taking these recommendations into consideration. which was not made public. No. however. 137. up to the time of its publication. reference 160). 107. the importance of specifications as an integral part of the risk assessment of food additives was stressed. the Committee had access to documents called Technical Data Sheets. At the fiftyninth meeting (Annex 1.2 Principles governing the toxicological evaluation of compounds on the agenda In making recommendations on the safety of food additives and contaminants.1 Modification of the agenda Flavouring agents Nos 909. which were prepared for new or existing food additives and which were not published because the detailed information on manufacturing processes described therein could be commercially sensitive. 116. including the present one. references 77. reference 76). 70 (EHC 70). the Secretariat has adapted the format and structure of the Technical Data Sheet and renamed it the Chemical and Technical Assessment (CTA).2. comments and recommendations made. also contain valuable information. 2 . The CTA reflects and emphasizes the role that chemical characterization plays in the risk assessment of food additives. 94. on chemical and technological aspects of the compounds under discussion. the Committee took into consideration the principles established and contained in Environmental Health Criteria. purity and use of the food additive. Environmental Health Criteria. as related to its risk assessment. 83. with the intention of making this document publicly available. 101. 149. Furthermore. These documents. contains the most important observations. Principles for the safety assessment of food additives and contaminants in food (Annex 1. the Committee recommended that these documents should include comprehensive information on technological use levels for foods. by the Committee and associated bodies in their reports on the safety assessment of food additives and contaminants. 2. 152 and 154).

1 FAO guidelines on the structure and content of the document called “Chemical and Technical Assessment (CTA)”: http://www. The use of such a substance.org/es/ESN/jecfa/guidelines1_en.The drafting expert responsible for preparing a CTA is asked to identify those sections of a confidential nature and the Secretariat will ensure that they are removed before publication. • There is a valid estimate of current exposure to the named substance and.stm 3 .2. or of the named chemical and identified secondary constituents. such that at least 95% of the commercially used material consists either of the named chemical. Some substances that have a use as flavouring agents may have been evaluated previously by the Committee in relation to other food additive functions. At its present meeting. including the generation of active flavouring substances during storage or processing of the food.fao. The Committee re-iterated the criteria that need to be met for an individual flavouring agent to be evaluated by the existing Procedure for the Safety Evaluation of Flavouring Agents: • The substance should be chemically defined. the Committee noted that a range of regulatory definitions of “flavouring” and similar terms exist in different countries and concluded that any definition would need to be elaborated in an international forum. it is not anticipated that they will be published in printed form. the Committee recognized the need for a working definition of the term “flavouring agent” and recommended that such a definition be agreed at a future meeting. At its present meeting. The CTAs will be available via the FAO JECFA website. the Committee reviewed the first set of CTA for certain food additives and provided feedback to the Secretariat on the FAO guidelines on the structure and content of the document called “Chemical and Technical Assessment (CTA)1”. its breakdown or reaction products. previously-established ADI.2 Safety evaluation of flavouring agents Working definition of “flavouring agent” At its fifty-ninth meeting. • The substance is added to food for flavouring purposes. such as the Codex Alimentarius Commission. 2. or its breakdown or reaction products. if appropriate. as a flavouring agent is included in the relevant.

4 . organizing the components of a natural flavouring complex into congeneric groups. more extensive data on the toxicity of these substances are required in order to complete their evaluation. evaluated by the “B-side” of the Procedure for the Safety Evaluation of Flavouring Agents At the present meeting. The revised Procedure builds on the Procedure for the Safety Evaluation of Flavouring Agents (Annex 1. dihydrocoumarin (No. 1172) that were evaluated by the Procedure for the Safety Evaluation of Flavouring Agents could not be predicted to be metabolized to innocuous end products (step B2) and their intake exceeded the human intake threshold for their structural class (step B3). In application of the Procedure. two flavouring agents. the Committee noted that the data required would include studies of metabolism and toxicity of the substance. several of which have been evaluated previously by the Committee. These studies would need to be of sufficient quality and duration to enable the flavouring agent to be evaluated at its specified intake. In considering such substances. or extraction with water or organic solvents. the Committee considered a working paper outlining a revision to the safety evaluation of flavouring agents to accommodate the safety evaluation of natural flavourings that are complex mixtures (natural flavouring complexes). These flavourings are obtained from a single source material by physical processes such as distillation. The Committee recommended that the guidelines for the preparation of monographs for flavouring agents be revised to ensure that a consistent approach is applied to the evaluation of such substances. The steps in the existing Procedure have been modified to accommodate the evaluation of congeneric groups and provide for an overall evaluation of the natural flavouring complex. The Committee noted that flavouring agents for which more extensive data were required should be clearly identified in the report of the meeting and that a complete description of the evaluation of such flavouring agents should be provided in the report item and the monograph. Safety evaluation of natural flavouring complexes At its present meeting. reference 131). Many natural flavouring complexes consist of mixtures of individual flavouring agents.Consideration of flavouring agents with high intakes. which become the focus of the safety evaluation. 1171) and 6-methylcoumarin (No. and that refined estimates of intake might additionally be needed. Data on structurally related substances could also be used to support the evaluation.

and the importance of ensuring that an inventory of commercial products be compiled was stressed. These include essential oils. The Committee noted that several issues being considered by this Project were of particular relevance to some of their present evaluations: 5 . The Committee also noted that criteria would need to be developed to elaborate specifications for natural flavouring complexes. For those substances with current usage in food. such as essential oils. The Committee considered that it was necessary to take account of the range of composition of natural flavouring complexes across all regions. The Committee noted that numerous products from different geographical regions are used as flavouring complexes. Since compositional data are required to complete a safety evaluation by the revised Procedure. the Committee recommended that a small number of natural flavouring complexes be evaluated by the revised Procedure at a future meeting. Intake data on flavouring agents The Committee discussed the data requirements for substances to be evaluated by the Procedure for the Safety Evaluation of Flavouring Agents. which are relatively well characterized in terms of their chemical composition. as well as extracts and oleoresins. 2. the Committee noted that several hundred natural flavouring complexes are currently in commercial use.In considering the revised Procedure. the Committee noted that further modification of the Procedure could be required for natural flavouring complexes that cannot be well characterized in terms of their composition. Flavouring agents without reported poundage data will not be evaluated by the Committee. The Committee was aware that different organizations have different approaches to the establishment of specifications for natural flavouring complexes. some of which are currently less well characterized.3 Joint FAO/WHO Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food The Committee was informed about the progress of this Project and recognized its importance. poundages used for intake assessments should be reported using no more than two significant figures. To determine the applicability of the revisions. The Committee concluded that the revised Procedure provides a potentially efficient way of evaluating natural flavouring complexes that are well characterized.

the Secretariat presented a project that had been proposed recently to FAO. e.1 Compendium of Food Additive Specifications and Guide At its forty-sixth and fifty-fifth meetings. non-progressive. particularly those of natural origin. At the present meeting. 2. — approaches for the development of specifications for complex mixtures. 2. Such advice may be elaborated by committees.— dose–response modelling of endpoints. reference 96) and the Guide to Specifications (Annex 1. which would consider the provision of scientific advice by both organizations to the Codex Alimentarius Commission and to Member countries. which cannot be assigned a threshold. reference 100). — revision of the approach to the safety evaluation of flavouring agents. integrity. efficiency and sustainability. ad hoc consultations or consultants. — relevance of reversible. for contaminants with longer biological half-lives. This consultative process is designed to improve the scientific advice provided with regard to quality. — biomarkers of effect and their relationships to disease outcome. treatment-related effects. for the development of a consistent. with the following objectives: 6 .5.g.5 Food additive specifications to Specifications 2. such as JECFA. both carcinogenic and non-carcinogenic. the Committee had recommended the revision of the Compendium of Food Additive Specifications (Annex 1. independence. timeliness. — longer tolerable intake periods. transparency.4 Provision of scientific advice by FAO and WHO The Committee was informed about a consultative process initiated by FAO and WHO. — probabilistic modelling for estimation of intake. provisional tolerable monthly intake (PTMI). The Committee noted that this exercise would take into consideration and build upon the experience of and the improvements already being implemented by the Secretariat of this Committee. The outcome of the process would be a set of recommendations. addressed to the Directors-General of FAO and WHO. harmonized and flexible overarching framework (an “umbrella”). The Committee was informed that Maria Lourdes Costarrica (FAO) and Wim van Eck (WHO) were responsible for the coordination of this consultative process. feasible and acceptable to all stakeholders. in order to accommodate natural flavours. which is realistic.

5. the Committee formulated a general method of analysis for residual solvents. It was also noted that a variety of gas chromatographic methods for the determination of residual solvents were included in the specifications. Depending on the availability of funds. since injectors for packed columns are no longer available. using head-space gas chromatography with flame ionization detection (FID). These methods should also respect the fact that they are applied by laboratories in developing and developed countries with varying levels of equipment and expertise.• The current edition of the Guide to Specifications will be updated and published together with a consolidated edition of the Compendium of Food Additive Specifications as one document in two volumes. This method is to be published in Section E of FNP 52. the project will start during 2003 and will terminate in 2005. The Committee noted that the General Method described in FNP 5 refers to obsolete gas chromatographs with packed columns. • The update shall reflect state-of-the-art analytical methodologies and practice by regulators and industry.2 Residual solvents Several of the specifications for food additives under review at the present meeting include limits for residual solvents. FNP 5 (Annex 1. and that it may be difficult to obtain such chromatographs. At its present meeting.5. The Committee concluded that specifications containing limits for residual solvents should refer to the same General Method in FNP 5 wherever possible. 2.3 Specifications of purity for flavouring agents The Committee agreed to replace the now outdated Council of Europe numbers with the recently introduced European Commission “FLAVIS database” numbers. reference 100). • The update shall consider the general guidelines laid out by this Committee and the Joint FAO/WHO Conference on Food Additives (summarized in EHC 70) and the work of other relevant standard-setting bodies • The update shall be available in print and electronically. In some cases. the methods of analysis to be used are included in the specifications and in others reference is made to the General Method included in the Guide to Specifications. The Committee recommended that FNP 5 be revised to include modern methodology. 2. 7 . Add 11.

Alternatively. When draft MLs are the only available information on additive use levels in food. the Guidelines indicate how to verify whether the assumptions on which the budget method is based are adequate. One of these tiers consists of combining estimated food intakes from various geographical regions with the draft proposed maximum levels (draft MLs) of additives for the Codex General Standard on Food Additives (GSFA).6 Intake assessment of food additives food additives 2.htm 8 . This example also illustrates that some food additives are listed in the GSFA for use in very broad food categories when in reality they are used in a very limited number of applications.2 Consideration of the Guidelines The Guidelines for the preparation of working papers on the intake of food additives were given further consideration and revisions were suggested by the Committee.6. However. In some cases. 1 JECFA Guidelines. In addition. are stressed. the estimation of the intake using high proposed GSFA levels has resulted in unrealistic estimates.1 Use of proposed maximum limits in the intake assessment of The Committee assesses dietary exposure to food additives using a tiered approach. 2. For example. in most cases. Consequently. at the present meeting the Committee evaluated annatto extracts and noted that they are used at a concentration of 35 mg/kg in Mimolette cheese.6.int/pcs/jecfa/jecfa_gls. the MLs in Codex standards are higher than the typical use levels reported by governments and industry. the limitations of marketshare data. these intake estimates were many times the corresponding ADI. an ML of 600 mg/kg is proposed in the GSFA for all cheese. as appropriate.who. The Committee observed that. the Committee suggests that the Codex Committee on Food Additives and Contaminants (CCFAC) might wish to review MLs with the aim of lowering them or restricting their use to food subcategories. according to the JECFA Guidelines1 . In the revised version. CCFAC may consider providing the Committee with typical use levels to allow for more realistic exposure assessments. http://www.2. and the need for data on levels of use by industry in order to make intake assessments.

The host strain was developed from a parent strain DN2717.3. The enzyme is subsequently partially purified and concentrated. LE399 a-amylase is produced by pure culture fermentation of a strain of B. “Termamyl LC”. The DN2717 strain was genetically engineered to inactivate the following native genes: the apr gene encoding the “Alkalase” protease. licheniformis that is non-pathogenic and non-toxigenic and which has been genetically modified to carry a genetically engineered gene coding for a-amylase. was introduced into the host strain SJ5550. Specific food additives (other than flavouring agents) The Committee evaluated six food additives for the first time and reevaluated a number of others. a derivative of a natural B. licheniformis isolate. LE399 a-amylase has not been evaluated previously by the Committee. The inactivated amyL. licheniformis 3. The engineered gene.1 Safety evaluations engineered a-amylase gene from B. The enzyme is thermostable and active at a relatively low pH and low calcium concentration.1 a-Amylase from Bacillus licheniformis containing a genetically The enzyme preparation under evaluation contains the enzyme LE399 a-amylase from the genetically modified Bacillus licheniformis. for example. sodium chloride. These characteristics make the enzyme particularly suitable for use in starch hydrolysis conducted at high temperatures. for the liquefaction of starch used in the production of nutritive sweeteners. the xyl gene encoding xylose isomerase. The a-amylase protein was developed by changing four amino acids in the polypeptide chain of another genetically engineered thermostable a-amylase. and glucose/ sucrose. and the gnt gene encoding gluconate permease. In the final preparation. xyl. 3. Information on the safety evaluations and on specifications is summarized in Annex 2. this LEC is stabilized and standardized/formulated with methionine.1. resulting in a liquid enzyme concentrate (LEC). designated as the LE399 a-amylase gene. These modifications were accomplished by introducing appropriate mutations into the DNA sequence encoding the Termamyl LC a-amylase. and gnt genes were replaced with three copies of the 9 . Details of further toxicological studies and other information required for certain substances are given in Annex 3. the amyL gene encoding the Termamyl a-amylase.

LE399 a-amylase gene. In a separate step, the gene encoding Ccomponent protease was deleted. The resulting strain was designated as MOL2083 and used as a production strain The aim of these genetic modifications was to produce the LE399 a-amylase, to prevent the synthesis of proteases that might hydrolyse the LE399 a-amylase, and to avoid the production of the Termamyl a-amylase. The genetic material introduced into the production strain has been well characterized and does not contain any sequences that would encode for proteins resulting in the production of toxic or undesirable substances. The LE399 a-amylase gene is stably integrated into the B. licheniformis chromosome. The production strain does not contain genes encoding proteins that inactivate antibiotics. The LE399 a-amylase was assessed for potential allergenicity by amino acid sequence comparison with known allergens listed in publicly-available protein databases. No immunologically-significant sequence homology was detected. Toxicological studies were conducted on the LEC. The materials added to the LEC for stabilization and formulation/standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns. In a 13-week study in rats, no significant treatment-related effects were seen when the LEC was administered by oral gavage at doses of up to and including 10 ml LEC/kg of body weight per day, the highest dose tested. Therefore this highest dose (equivalent to 1.02 g total organic solids (TOS)/kg of body weight per day) was considered to be the NOEL. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and was not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro. The a-amylase preparation is intended for use in starch liquefaction in the production of sweetener syrups, alcoholic beverages and beer. The absence of the a-amylase protein in the final (purified) sweetener syrup has been confirmed experimentally. In the spirits industry, no LE399 a-amylase or other organic solids are expected to be carried over to the final product because ethanol is removed by distillation from the fermentation mash containing the enzyme preparation. In the brewing of beer, the enzyme preparation is added during the mashing process and is denatured and inactivated during the subsequent wort-boiling stage. The beer filtration process is likely to remove the denatured enzymes along with other insoluble materials. In conclusion, no residual LE399 a-amylase is expected to be present in food processed using this enzyme preparation.
10

Nevertheless, very conservative estimates of daily intakes were performed on the assumption that all the TOS would persist in the final products, giving an estimated daily intake of 12 mg TOS/day (equivalent to 0.2 mg TOS/kg of body weight per day) for sugar and syrups, 3 mg TOS/day (equivalent to 0.05 mg TOS/kg of body weight per day for a 60 kg person) for beer and 10.8 mg TOS/day (equivalent to 0.18 mg TOS/kg of body weight per day) for spirits. Compared to the NOEL of 1020 mg TOS/kg of body weight per day derived from the 13-week study of oral toxicity, the margin of safety is >2000. The Committee allocated an ADI “not specified” to a-amylase from this recombinant strain of B. licheniformis, used in the applications specified and in accordance with good manufacturing practice. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established.
3.1.2 Annatto extracts

Annatto extracts have been used for over two centuries as a food colour, especially in cheese, and various types are now used in a wide range of food products. Annatto extracts are obtained from the outer layer of the seeds of the tropical tree Bixa orellana. The principal pigment in annatto extract is cis-bixin, which is contained in the resinous coating of the seed itself. Processing primarily entails the removal of the pigment by abrasion of the seeds in an appropriate suspending agent. Traditionally, water or vegetable oil is used for this purpose, although solvent extraction is also employed to produce annatto extracts with a higher pigment content. Microcrystalline bixin products of 80–97% purity have been developed in response to the need for more concentrated annatto extracts. Annatto extracts were evaluated by the Committee at its thirteenth, eighteenth and twenty-sixth meetings (Annex 1, references 19, 35, 59– 61). At its eighteenth meeting, the Committee considered the results of long-term and short-term tests in experimental animals fed an annatto extract containing 0.2–2.6% pigment expressed as bixin. A longterm study in the rat provided the basis for evaluation; the NOEL in this study was 0.5% in the diet, the highest dose tested, equivalent to 250 mg/kg of body weight. A temporary ADI was established of 0– 1.25 mg annatto extract/kg of body weight. The Committee re-evaluated annatto extract at its twenty-sixth meeting, when the results of the metabolic studies that had been requested became available. Studies of mutagenicity, additional long-term
11

(1-year) studies in the rat, and observations of the effects of annatto extract in humans were also considered. No evidence was found for the accumulation of annatto pigments in the tissues of rats fed at low concentrations (20–220 mg/kg of body weight per day of annatto extracts containing up to 2.3% bixin/norbixin mixture) for a year. Studies in both rats and humans showed that although annatto pigments are absorbed from the intestine into the blood, clearance from the plasma is rapid. The NOEL in the original long-term rat study was determined as 0.5% in the diet, equivalent to 250 mg/kg of body weight, and the ADI was set at 0–0.065 mg/kg of body weight of annatto extract expressed as bixin. In this re-evaluation, the Committee considered the highest concentration of bixin in the material tested (i.e. 2.6%) and established an ADI on the basis of the content of bixin. B. orellana is grown in many countries and various procedures are used to produce annatto extracts from the seeds for commercial use. The following extracts were considered for evaluation:
Annatto extract (solvent-extracted bixin): Annatto B1

The seeds are extracted with solvent to dissolve the pigment. The extract is filtered to remove insoluble material. Subsequent processing involves removal of fats and waxes, solvent removal, crystallization and drying.
Annatto extract (solvent-extracted norbixin): Annatto C

The seeds are extracted with solvent to dissolve the pigment. The extract is filtered to remove insoluble material. Subsequent processing involves removal of fats and waxes, removal of the solvent, crystallization and drying. Aqueous alkali is added to the resultant powder, which is heated to hydrolyse the pigment and then cooled. The aqueous solution is filtered, and acidified to precipitate the norbixin. The precipitate is filtered, washed, dried and milled to give a granular powder.
Annatto extract (oil-processed bixin suspension): Annatto D

The seeds are abraded in hot vegetable oil to remove the pigment.

1

To ensure clarity, the Committee adopted the designations B, C, D, E, F, G, as employed in the submitted information, to refer to the different annatto extracts under evaluation

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Annatto extract (alkali-processed norbixin): Annatto F The seeds are abraded in cold aqueous alkali (potassium or sodium hydroxide) to remove the pigment. washed. The new studies consist primarily of 28-day and 90-day studies. Additional alkali is added to the resultant suspension. dried and milled to give a granular powder. These preparations are traded between primary processors of annatto seeds and the final vendors of colour products to the food companies. Additional alkali is added to the resultant suspension. studies of effects on microsomal oxidation enzymes and studies of genotoxicity with these formulations. 13 . The pigment content. At its present meeting. not acid-precipitated): Annatto G The seeds are abraded in cold aqueous alkali (potassium or sodium hydroxide) to remove the pigment. solvent-extracted annatto extract containing 91. The non-pigment fractions of the concentrated extracts are not well characterized.Annatto extract (aqueous-processed bixin): Annatto E The seeds are abraded in cold aqueous alkali (potassium or sodium hydroxide) to remove the pigment. of which 97% was bixin and 1. dried and milled to give a granular powder.6% norbixin. the main pigments contributing to the colour of annatto extracts. The resulting suspension is acidified to precipitate the bixin. The aqueous solution is filtered.7% norbixin. expressed as bixin and norbixin. of the extracts considered for evaluation is as follows: Annatto B: Annatto C: solvent-extracted annatto extract containing 92% pigment. too concentrated to add directly to foods and require dilution with carriers such as vegetable oil (with emulsifiers). Fats and waxes are removed. Bixin and norbixin. washed. however. They are. Potassium carbonate may be added. propylene glycol or alkali. disposition studies. The aqueous solution is filtered. Annatto extract (alkali-processed norbixin. and then cooled. which is heated to dissolve the pigment and then cooled. and dried. are present at different concentrations in different commercial preparations. Fats and waxes are removed. the Committee evaluated new studies involving several concentrated preparations containing bixin and norbixin. which is heated to dissolve the pigment. and acidified to precipitate the norbixin. The precipitate is filtered. one study of developmental toxicity and data on the potential allergenicity of annatto extract. The precipitate is filtered.

01%) after administration of Annatto E. suggesting that norbixin is not converted to bixin in the body. Bixin and norbixin are mostly cleared from the plasma within 24 hours. the concentrations of norbixin in blood were higher than those of bixin and persisted for longer.8% pigment. of which 94% was bixin and 1. of which 90% was bixin and 4.7% norbixin.5 mg norbixin. Cis-norbixin appears to be readily converted to trans-norbixin. It is unclear whether conversion of bixin to norbixin occurs in the body.Annatto D: Annatto E: Annatto F: Annatto G: oil-processed annatto extract containing 10.1% norbixin. The presence of norbixin in plasma after administration of Annatto B and E suggests that bixin may be converted to norbixin in the body. No bixin was detected in the urine following administration of any of the annatto extracts. Bixin was not detected in plasma after oral administration of norbixin to rats. since bixin could not be detected in the plasma 8 hours after a single oral dose whereas norbixin reached a peak after 4 hours and was still detected after 48 hours. C.2% norbixin. E and F. but these preparations also contain norbixin which could have accounted for the norbixin levels in plasma. In humans given a commercial preparation containing 16 mg bixin and 0. The new data confirmed earlier findings that there appears to be at least partial absorption of bixin and norbixin. The total percentage of the dose of bixin and norbixin absorbed cannot be determined from the available data. Although studies have shown that about half of the administered dose appears in the faeces. alkali-processed annatto extract containing 41. The more polar acid norbixin is absorbed to a greater extent than the less polar bixin. New toxicological data were made available for four of these extracts: Annatto B. sodium and potassium salts containing 17.5% norbixin. Toxicological studies. 14 . and that the colours in water-soluble preparations of annatto are more readily absorbed than the oil-soluble preparations. and traces (<0. aqueous-processed annatto extract containing 26% pigment. alkali-processed annatto extract. it is not possible to determine whether this is because the pigments are not absorbed and pass through the gastrointestinal tract. or whether part of the dose is absorbed and is excreted via the bile. but extremely small amounts of norbixin (<3% of the dose) were found in urine after administration of Annatto F.

All three annatto extracts induced CYP4A. Annatto F caused the greatest induction of CYP4A and there was an increase in the number of mitochondria observed by electron microscope with Annatto C. Studies in mongrel dogs fed a chloroform-extracted preparation of annatto in glucose and similar studies in rats and mice fed an ethanolextracted preparation of annatto. but in 15 . Weak positive results at toxic concentrations were noted for some preparations in tests for mutagenicity in mammalian cells in the absence of an endogenous metabolic activation system. particularly in male rats. E or F) revealed that Annatto B and E are inducers of CYP1A2. There was no evidence that any of the annatto extracts was a phenobarbital-type inducer. The absence of any further hypertrophy between days 28 and 90 of treatment. the Committee concluded that annatto extracts are not carcinogenic. Since the results of tests on analytical grade bixin and norbixin were negative. or was an inducer of CYP2E1. The pattern of induction of CYP1A2 and CYP4A by different preparations in different sexes indicate that these effects are independent. This conclusion was based on the results of tests with annatto preparations containing low concentrations of bixin. and the absence of pathological changes in the liver. E and F preparations did not demonstrate any potential to cause genetic damage in a test for micronucleus formation in bone marrow in vivo.Examination of cytochrome P450 (CYP450) enzymes in liver samples at the end of 10-week studies in rats fed with one of several annatto preparations (Annatto B. In vitro studies of genotoxicity revealed equivocal and inconsistent positive results only at concentrations that exceeded solubility or at concentrations that were cytotoxic. were considered not to be relevant to the extracts being evaluated. whereas weak positive results were noticed only in the presence of an endogenous metabolic activation system in tests for chromosomal aberration. Results of tests for mutagenicity in mammalian cells and for chromosomal aberration were inconsistent. There were only slight increases in CYP3A1 and CYP3A2. observations which are consistent with the action of peroxisome proliferators. No new studies of carcinogenicity have become available. may be compatible with metabolic adaptation. Liver weight increases were not related to the increase in cytochrome P450 enzymes. Studies in mice receiving Annatto B. some weak positive results obtained with the concentrated annatto extracts in bacterial tests in the absence of an endogenous metabolic activation system were considered to be caused by other components in the annatto preparations. In its previous evaluations.

as only non-specific toxicity was reported at the higher doses tested. which was accompanied in some cases by centrilobular hepatocellular hypertrophy. corresponding to 1170 mg or 1290 mg of bixin/kg of body weight per day. E and F. The NOEL for Annatto B was identified as 16 000 mg/kg diet (equal to 1311 mg and 1446 mg of extract/kg of body weight per day for males and females respectively.a study of the initiation/promotion of liver carcinogenesis. haematological changes and alterations in serum proteins. The NOEL for Annatto C was identified as 1000 mg/kg diet (69 mg and 76 mg/kg of body weight per day for males and females respectively. corresponding to 172 mg or 180 mg of bixin/kg of body weight per day. Together with the results of the tests for genotoxicity and the absence of proliferative lesions in the short-term tests for toxicity. it is not clear whether the non-specific toxicity was attributable to bixin or norbixin or to other components present in the extracts. A common feature was the increase in absolute and relative liver weights at high and intermediate doses. and 21 mg or 23 mg of norbixin/kg of body weight per day) on the basis of urinary effects (elevated concentrations of protein in urine and crystals in urine sediment). The NOEL for Annatto E was identified as 10 000 mg/kg in the diet (734 mg and 801 mg/kg of body weight per day for males and females respectively. corresponding to 33 mg and 36 mg of norbixin/kg of body weight per day) on the basis of increased kidney weights. C. The NOEL for Annatto F was identified as 1000 mg/kg in the diet (79 mg and 86 mg/kg body weight per day for males and females respectively. A study of developmental toxicity in rats fed an annatto extract with a bixin content (28%) comparable to that of Annatto E at doses of up to 500 mg/kg of body weight per day (equal to 140 mg of bixin/kg of body weight per day) confirmed the absence of developmental toxicity at this dose. have low toxicity. Annatto C did not increase the incidence of preneoplastic lesions. Studies of subchronic toxicity demonstrated that the annatto extracts tested. 16 . and 8 mg or 8.8 mg of norbixin/kg of body weight per day) on the basis of increases in thyroid and kidney weights and decreased spleen weights. corresponding to 63 mg or 70 mg of norbixin/kg of body weight per day) on the basis of increases in liver weight accompanied by hepatocellular hypertrophy and necrosis. As is the case with genotoxicity. Annatto B. this is supportive of earlier conclusions.

40 mg/day. A revision of the previous intake estimate was performed on the basis of typical use levels of extracts expressed as bixin and norbixin provided by industry.4 mg/kg bw (based on NOELs of 69 mg and 76 mg/kg of body weight per day in male and female rats respectively). An additional safety factor of 2 was applied to the NOELs. Comparison of the estimated intakes with the temporary ADI values were performed assuming that each annatto extract was a unique 17 . These levels were combined with various national intake data. Other components in the extracts might contribute to or be responsible for the effects noted. on the basis of data from the United Kingdom.0 mg/kg of body weight (based on NOELs of 734 mg and 801 mg/kg of body weight per day in male and female rats respectively).03 mg and 0. With the application of a 200-fold safety factor to the NOEL for each of the annatto preparations.4 mg/kg of body weight (based on NOELs of 79 mg and 86 mg/kg of body weight per day in male and female rats respectively). because of deficiencies in the database. The resulting average intake is observed to be between 0. but the results of oral challenges were inconclusive because of inadequate study design or lack of statistical significance.The differences in NOEL for Annatto B (bixin) and Annatto C (norbixin) indicate that extracts that mainly contain norbixin are more potent than those containing mainly bixin. Estimated intake for high consumers reaches 1. A number of studies on possible allergic potential in humans were available. Annatto F: 0–0. the following temporary ADIs were allocated: Annatto B: 0–7. and no ADI could be established. The potencies of the different extracts cannot be explained on the basis of their bixin and/ or norbixin contents. The Committee could not establish a generic ADI for the various annatto extracts on the basis of the data submitted and therefore established a temporary ADI for each of the individual preparations tested.0 mg/kg of body weight (based on NOELs of 1311 mg and 1446 mg/kg of body weight per day in male and female rats respectively). Evaluation. Annatto C: 0–0. Annatto E: 0–4. No data on the potential toxicity of Annatto D or Annatto G were available.50 mg/day. and/or the differences in potency might have arisen from differences in bioavailability of the extracts.

Specifications. 3.6-diene-3.1. The Committee requested additional information to clarify the role that the non-pigment components of the extract play in the expression of the qualitative and quantitative differences in toxicity of the various extracts. i. with purification of the resultant extract by crystallization. The principle purpose of specifications is to ensure that the material of commerce is comparable to the material that has been biologically tested.5-dione) and its desmethoxy and bisdesmethoxy derivatives in varying proportions.7-bis(4-hydroxy-3methoxyphenyl) hepta-1. In addition. which had not been tested biologically. (C. 18 . that contains norbixin. The commercial product consists essentially of curcumins: the colouring principle (1. such as Annatto F. This Committee adopted tentative specifications for the four annatto extracts tested. the Committee requested data on the reproductive toxicity of an extract. the ground rhizomes of Curcuma longa L.source of bixin/norbixin. of which not more than 2. These simulations show in each case that the estimated exposure for adults is <20% of the corresponding temporary ADI. A toxicological monograph and a chemical and technical assessment (CTA) were prepared.3 Curcumin The food colour curcumin (turmeric yellow) is obtained by solvent extraction of turmeric. domestica Valeton).5% is norbixin) Annatto extract (solvent-extracted norbixin) — Annatto C: not less than 85% pigment (as norbixin) Annatto extract (aqueous-processed bixin) — Annatto E: not less than 25% pigment (as bixin.e. with the following minimum assay values: Annatto extract (solvent-extracted bixin) — Annatto B: not less than 85% pigment (as bixin. of which not more than 7% is norbixin) Annatto extract (alkali-processed norbixin) — Annatto F: not less than 35% pigment (as norbixin) The Committee also adopted tentative specifications with minimum assay values as proposed for the commercial products Annatto D and G. Minor amounts of oils and resins that occur naturally in turmeric may also be present. The total content of colouring matter (curcuminoids) in curcumin is not less than 90%.

the Committee evaluated the results of studies of carcinogenicity in rats and mice given turmeric oleoresin containing 79–85% curcuminoids. The Committee again extended the temporary ADI. twenty-second. On the basis of the NOEL of 220 mg/kg of body weight per day for liver enlargement observed in the study of carcinogenicity in mice.1 mg/kg of body weight for curcumin. the product of solvent extraction of turmeric containing <90% of total colouring matter (curcuminoids). Turmeric oleoresin. forty-fourth. whereas the ADI for turmeric oleoresin was withdrawn at the thirty-fifth meeting. twenty-fourth. and new biochemical and genotoxicity data.5 mg/kg of body weight) and an assumed average concentration of 3% curcuminoids in turmeric. thirtieth. 53. thirty-ninth. pending submission of the results of a study of reproductive toxicity with curcumin to be reviewed in 1998. 88. twenty-sixth.The term “curcumin” in this report refers to the material for which specifications exist. The low survival rate of pups in the mouse study and the low rates of pregnancy in rats led the Committee to conclude that these studies did not provide assurance that the potential reproductive effects of curcumin had been adequately investigated. 1. The temporary ADI for curcumin was extended at the twenty-second. At its forty-fourth meeting. and a safety factor of 200.7-bis(4hydroxy-3-methoxyphenyl) hepta-1.5% curcuminoids). At its fifty-first meeting. is often referred to as curcumin in the literature. 101. the Committee requested the results of studies of carcinogenicity in mice and rats fed turmeric oleoresin and the results of a study of reproductive and developmental toxicity associated with curcumin. At the thirty-ninth meeting.0–76. references 19. 73. the Committee established a temporary ADI of 0–0. 35. twenty-sixth. 116. 59.5-dione. the Committee evaluated the results of studies of fertility in rats and mice treated with turmeric oleoresin (68.6-diene-3. thirtieth. At its eighteenth meeting. thirty-fifth and thirty-ninth meetings. and this name will be used below when it is necessary to refer to the principal colouring component of curcumin. the Committee decided that this report would not use the term curcumin when referring to this substance. based on the then existing ADI for turmeric oleoresin (0–2. thirty-fifth. A common synonym for this substance is diferuloylmethane. and curcumin were evaluated by the Committee at its thirteenth. to avoid confusion. twenty-fourth. eighteenth. pending 19 . 137 and 154). fifty-first and fifty-seventh meetings (Annex 1. 47. the Committee increased the temporary ADI to 0–1 mg/kg of body weight and extended it. The principal colouring component. The Committee concluded that data on developmental toxicity were no longer required but reiterated its request for a study of reproductive toxicity.

at the intermediate and high doses. and 0. or at high risk of cancer. 14 and 21 at the high dose. pup survival or fertility indices in either generation. for review in 2001. The total period of treatment was 21 weeks for the parental generation and 24 weeks for the F1 generation. This change was therefore considered to be incidental. The changes seen at the high dose (equal to 960–1100 mg/kg of body weight per day for the F1 parental generation) could be an indication of a persistent decrement in body-weight gain. In addition. There were no significant differences in maternal body weights at the end of the gestation period and no adverse effects were observed in the F1 offspring. Diferuloyl20 . Wistar rats were fed diets containing curcumin (comprising 80% diferuloylmethane and 99% total curcuminoids) at doses equal to 0. body weight.3%) doses of >2000 mg/day (>33 mg/kg of body weight per day.submission of the results of a study of reproductive toxicity with a substance complying with the specifications for curcumin. and days 7. There were no other effects on general health. The results of the multigeneration study were available to the Committee for evaluation at the current meeting. for a 60 kg adult). diferuloylmethane could be detected in plasma after oral diferuloylmethane (99. In pharmacokinetic studies in these patients. The Committee therefore concluded that the NOEL for decreased pup body weight was 250 mg of diferuloylmethane/kg of body weight per day. At its fifty-seventh meeting. The effect on pup weight seen at the intermediate dose (equal to 250–320 mg/kg of body weight per day) was likely to be incidental. Biological data. Two clinical trials were conducted in patients with cancer. 250–290 and 850–960 mg/kg of body weight per day in males. but not after lower doses. the Committee was informed that a multigeneration study in the rat was in progress. Significant decreases in the average weights of the F2 generation pups were observed at days 1 and 7 at the intermediate dose. 160. 130–140. and thus extended the temporary ADI of 0–1 mg/kg of body weight until 2003. 310–320 and 1000–1100 mg/kg of body weight per day in females. the Committee reviewed the results of two new clinical trials investigating either an extract of Curcuma or diferuloylmethane as potential anticancer agents. but not the F1 generation. Transient minor decreases in maternal body-weight gain were observed during gestation days 10–15 in the parental. These decrements represented <10% of the average weight of the concurrent controls and were reported to be within the range of the historical control data. In the multigeneration study of reproductive toxicity.

methane. and the application of a safety factor of 100.3%) at doses of up to 8000 mg/day for 3 months. The new multigeneration study in rats that were fed with curcumin for periods of up to 24 weeks met the Committee’s requirements. Therefore the Committee concluded that adequate data were not available to accurately assess the exposure. In the course of the study. These clinical trials provided limited information of relevance to the assessment of toxicity of curcumin. Taking into account all of the data evaluated previously. Fifteen patients receiving an extract of Curcuma (18 mg of diferuloylmethane and 2 mg of the desmethoxy derivative suspended in 200 mg of essential oils derived from Curcuma spp. is an unrealistic overestimate of the exposure. The report stated that FSANZ was not able to provide national estimates of intake for Australia or New Zealand owing to the regulatory status of curcumin. which is allowed at levels consistent with good manufacturing practice in all foods. This estimate. The Committee noted that the previous temporary ADI was derived from a study on turmeric oleoresin (79–85% curcuminoids) that did not comply with the current specification. one patient (receiving 108 mg/day diferuloylmethane) experienced nausea and two patients (receiving 72 and 180 mg/day diferuloylmethane) experienced diarrhoea. on the basis of the NOEL of 250–320 mg/kg of body weight per day in the multigeneration study in rats. this material met the specification developed at this meeting. There were no reported adverse effects in a study of twenty-five patients taking diferuloylmethane (99. combining maximum curcumin use levels from the draft General Standard for Food Additives (GSFA) with food consumption data. Additionally.) at daily doses of 26–180 mg of diferuloylmethane for up to 4 months. An addendum to the toxicological monograph and a chemical and technical assessment (CTA) were prepared. The Committee received an estimate of intake only from Food Standards Australia New Zealand (FSANZ). was detected in the faeces but not in the urine. were monitored for adverse effects by physical examination and tests for haematological parameters. 21 . The Committee considered that these ancillary studies could not be used to derive an ADI for curcumin. the NOEL was 250–320 mg/kg of body weight per day. and in one patient diferuloylmethane sulfate. Assessment of intake. the Committee withdrew the temporary designation and allocated an ADI of 0–3 mg/kg of body weight for curcumin. Decreased weight gain in the F2 generation was observed at doses equal to 960–1100 mg/kg of body weight per day of curcumin.

the Committee established specifications for both the above-mentioned products under the name “diacetyltartaric and fatty acid esters of glycerol”. is recognized as an extraction solvent.Specifications. with the exception of adrenal medullary tumours (designated as phaeochromocytomas) in male 22 . citric. pending submission of additional information concerning adrenal medullary and cardiac lesions observed in the 2-year study in rats. Higher incidences of these lesions were observed in all groups in the reassessment compared to the earlier analysis.1. 3. Because carbon dioxide is a gas at ambient conditions.4 Diacetyltartaric and fatty acid esters of glycerol Diacetyltartaric and fatty acid esters of glycerol (DATEM) were reviewed by the Committee at its tenth and seventeenth meetings (Annex 1. the Committee also reviewed fatty acid esters of glycerol with acetic. as the Committee was aware that the two products could not be distinguished analytically. as a supercritical fluid. the Committee allocated an ADI of 0–50 mg/kg of body weight on the basis of the results of studies of biochemical and metabolic parameters and tests in animals receiving DATEM in the diet. A residual limit for ethyl acetate of 50 mg/kg was included in the specification monograph. reference 137). At that meeting. Ethyl acetate has been evaluated previously by the Committee as a carrier solvent. Ethyl acetate and carbon dioxide were added as alternative solvents. lactic and tartaric acids and allocated a collective ADI “not limited” to this group. the Committee recommended that the material defined in the specifications be evaluated toxicologically. New data were evaluated at the fifty-seventh meeting and the previous ADI of 0–50 mg/kg of body weight was made temporary. At its fifty-first meeting (Annex 1. At its seventeenth meeting. Carbon dioxide. At the present meeting. At the same meeting. No differences in survival between the control and DATEM-treated groups were reported. references 13 and 32). no limit for residual carbon dioxide is needed in this case. with the provision that the intake of tartaric acid should not exceed 30 mg/kg of body weight per day. The existing specifications were revised. Significant increased incidences of both lesions in the group treated with a high dose of DATEM and in the reference control group receiving an equivalent quantity of monoglyceride had been noted in this study. the Committee considered information provided on the reassessment of the histopathological data related to the adrenal and cardiac lesions from the 2-year study in the rat.

and with the provision that the total intake of tartaric acid from food additives should not exceed the ADI for tartaric acid (0– 30 mg/kg of body weight). The Committee considered that the NOEL for DATEM was 10% in the diet (equal to 4100 mg/kg of body weight per day in males and 6100 mg/kg of body weight per day in females) corresponding to the highest dose tested in the 2-year combined study of toxicity/carcinogenicity in the rat. This lesion was considered to reflect an earlier step in the process leading to myocardial fibrosis. At its fifty-fifth meeting. the results of a study in volunteers and some publications concerning the increased uric acid concentrations in serum after intake of d-tagatose. humectant. in the groups receiving 10% reference substance (monoglycerides) or 10% DATEM compared with untreated controls.75 g/kg of 23 . Its properties permit its use as a bulk sweetener. No difference in the incidence of myocarditis was observed between the reference control group and the group receiving 10% DATEM. references 149 and 154). texturizer and stabilizer. the Committee evaluated the results of four studies in experimental animals. D-Tagatose was evaluated by the Committee at its fifty-fifth and fifty- seventh meetings (Annex 1. There was an increase in the incidence of an inflammatory lesion. the Committee concluded that d-tagatose was not genotoxic.5 D-Tagatose D-Tagatose is a ketohexose. A NOEL of 0. embryotoxic or teratogenic. It also concluded that an ADI could not be allocated for d-tagatose because of concern about its potential to induce glycogen deposition and hypertrophy in the liver and to increase the concentrations of uric acid in serum. The Committee removed the temporary designation and allocated an ADI of 0–50 mg/kg of body weight. The results of the reassessment indicated that 2 years of treatment with DATEM did not affect the incidence of myocardial fibrosis. The incidence of adrenal medullary hyperplasia and adrenal medullary tumours in DATEM-treated groups was not higher than in untreated controls. 3. The Committee decided to base its evaluation on the human data reviewed in the course of the two meetings. myocarditis. It is obtained from D-galactose by isomerization under alkaline conditions in the presence of calcium. an epimer of D-fructose inverted at C-4.rats. other sugars.1. on the basis of the established NOEL with the application of a 100-fold safety factor. and other food components. At its fifty-seventh meeting.

Two new human studies have shown that a single dose of 30 g dtagatose to small numbers of healthy volunteers. Studies of d-tagatose administered to rats in the diet. the administration of diets containing 2. 20% fructose. and that Sprague-Dawley rats were more sensitive to these effects than Wistar rats. kidneys and testes. and no recorded gastrointestinal effects. the testes in males. At its forty-eighth meeting. Increased absolute and relative adrenal weights were observed in female rats at all doses of d-tagatose. in particular. the Committee noted that dfructose increases uric acid production by accelerating the degradation of purine nucleotides. was not specifically investigated. or 10% d-tagatose plus 10% fructose did not result in histological changes in the liver. at least in part. The degradation of d-tagatose-1-phosphate is slower than 24 . but not in those receiving fructose alone. In the absence of histopathological confirmation of the nature of the changes induced by d-tagatose in the adrenals. The weights of the kidneys in females. however. confirming the previous observation of strain differences. In a 2-year study in Wistar rats. increased liver weight and hypertrophy. probably by hepatocellular depletion of inorganic phosphate resulting from accumulation of ketohexose-1phosphate. on 5% d-tagatose. reviewed previously by the Committee. by glycogen accumulation. Increased adrenal weights were also reported in male rats fed on 5% and 10% d-tagatose.body weight per day was identified from a 28-day study in which no effects were observed in humans receiving three doses of 15 g of d-tagatose per day. focused on the hepatic effects of d-tagatose. or 15 g d-tagatose to hyperuricaemic individuals. and the caecum in each sex were also increased in animals fed on 10% d-tagatose. of six rat strains. and the smallest increase occurred in Wistar rats. the largest increase in liver weight occurred in Sprague-Dawley rats. At the present meeting. These studies indicated that these effects were caused. it is not possible to assess their toxicological significance to humans. and in some cases. the Committee reviewed the results of two new studies of toxicity conducted in rats. 5 or 10% d-tagatose.5. had no biologically significant effect on uric acid production or excretion. An ADI of 0–80 mg/kg of body weight for dtagatose was established on the basis of this NOEL and a safety factor of 10. and of two new studies of concentrations of plasma uric acid in human volunteers. The role of glycogen. although increased liver weights were reported in male and female rats fed on 10% d-tagatose. The new 28-day study investigating the effects of 20% d-tagatose in the diet has shown that.

25 . The study on hyperuricaemic individuals discussed at the current meeting indicated that the NOEL is also applicable to this vulnerable group. hyperuricaemic individuals are therefore potentially vulnerable to the adverse effects of d-tagatose. the maximum increases in serum uric acid and d-tagatose and the maximum decrease in serum ATP were seen within one hour of ingesting d-tagatose. Pending provision of the histopathology data. kidneys and testes of the rats from the 2-year study. The previous ADI was thus removed. but this could not be confirmed in the absence of histopathological examination of these tissues. and thus addressed concerns expressed at the fifty-fifth meeting. the Committee confirmed that the human data provided the most relevant basis for assessing the acceptable intake of d-tagatose. The new study demonstrated no increase in serum concentrations of uric acid within 4 hours of consumption of 15g of d-tagatose by this vulnerable group. the Committee identified a NOEL for healthy individuals of 45 g d-tagatose per day in three divided doses. and a safety factor of 6. this study also identified new effects. The Committee considered that these changes might have been due to high osmotic load resulting from the high dietary doses administered.that of d-fructose-1-phosphate. the Committee concluded that the ADI should be temporary and applied an additional safety factor of 2.6-diphosphatase. namely increased adrenal. kidneys and testes in the 2-year study in rats. kidney and testes weights. and on the basis of the NOEL of 0. the Committee allocated a temporary ADI of 0– 125 mg d-tagatose/kg of body weight. However.75 g/kg of body weight per day. and therefore the hyperuricaemic effect of d-tagatose may be greater than that of d-fructose. In view of the additional uncertainty regarding the nature of the effects observed in the adrenals. The Committee requested information on the histological examination of the adrenals. In studies reviewed previously by the Committee. The temporary ADI does not apply to individuals with hereditary fructose intolerance caused by deficiency in 1-phosphofructoaldolase (aldolase B) or fructose 1. At the fifty-seventh meeting. It is therefore anticipated that no effect would be observed in hyperuricaemic individuals following repeated consumption of 15 g of d-tagatose at subsequent meals. The Committee considered that a safety factor of 3 would be appropriate to allow for interindividual variation. The Committee concluded that the results of the 2-year study in rats established that the previously-reported liver glycogen deposition and hypertrophy observed after long-term administration of d-tagatose did not result in histopathological changes. by 2006.

1. This production strain was obtained from A. proteins. oryzae production strain.WT and pToC90. designated as Mt. glycine. and alcohols to form off-flavour precursors. potassium sorbate. the enzyme is partially purified and concentrated. and sodium benzoate. After fermentation. a promoter and a terminator. The pToC90 plasmid also contains the bla gene. The laccase gene is linked to DNA regulatory sequences. Laccase is an enzyme that catalyses the oxidation of phenolic compounds such as ortho.6 Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae The enzyme preparation under evaluation contains the active enzyme laccase. 26 . amino acids. this LEC is stabilized and standardized/formulated with sorbitol.WT plasmid contains the laccase gene from the thermophilic fungus. An addendum to the toxicological monograph and a chemical and technical assessment (CTA) were prepared. oryzae to metabolize acetamide in the absence of other sources of carbon or nitrogen and which is used as a selection marker. that is found in decaying organic matter. resulting in a liquid enzyme concentrate (LEC). was developed by transfection of the A. The pRAMB17. The pRaMB17.and para-diphenols to their corresponding quinones.WT plasmid also contains the bla gene. The laccase evaluated by the Committee is produced by pure culture fermentation of a strain of Aspergillus oryzae that is non-pathogenic and non-toxigenic and which has been genetically modified to carry a gene coding for laccase derived from Myceliophthora thermophila.The intake assessment prepared by the Committee at its fifty-seventh meeting is still valid. which has not been evaluated previously by the Committee. The pToC90 plasmid contains the amdS gene that encodes acetamidase. and other well-characterized DNA sequences. Laccase scavenges oxygen which otherwise would react with fatty acids. In the final preparation. oryzae host strain How B711 (derived from the A 1560 strain) using recombinant DNA techniques and traditional mutagenesis. pRaMB17. with the concomitant reduction of oxygen to water. The existing specifications were maintained 3. This enzyme is marketed for use in the brewing of beer to prevent the formation of off-flavour compounds. which confers resistance to ampicillin. thermophila. The A. such as trans-2-nonenal. which enables A. glucose. M. sodium lactate. oryzae host strain How B711 (derived from the A 1560 strain) with two plasmids.

Studies of skin and eye irritation in rabbits did not reveal treatment-related effects.e. The beer filtration process is likely to remove the denatured enzymes along with other insoluble materials. The genetic material introduced into the production strain has been well characterized using known molecular biology methods and does not contain any sequences that would encode proteins that are toxic or that produce toxic or undesirable substances. Thus no residual LE399 a-amylase is expected to be present in food processed using this enzyme preparation. no significant treatment-related effects were seen when the LEC was administered by oral gavage at doses of up to and including 10 ml LEC/kg of body weight per day. 27 . The materials added to the LEC for stabilization and formulation/standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns. thermophila laccase enzyme was assessed for potential allergenicity by amino acid sequence comparison with allergens listed in publicly available protein databases. No bla DNA was detected in the laccase preparation.The selection of transformants was achieved by growing on a medium containing acetamide as the sole nitrogen source and screening for ability to produce laccase. this gene is not expressed because it is under the control of a bacterial promoter that is not functional in the eukaryotic fungus A. Therefore this highest dose tested (equivalent to 1700 mg total organic solids (TOS)/kg of body weight per day) is considered to be the NOEL. the enzyme b-lactamase that hydrolyses and inactivates ampicillin. The M. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro. The LEC did not show skin sensitizing potential in a repeated patch test in humans. Furthermore. Although the introduced DNA contains the bla gene. i. No immunologicallysignificant sequence homology was detected. In the brewing of beer. the bla gene is stably integrated into the host genome. Thus. the laccase preparation does not contain the bla gene product. oryzae genome. the laccase preparation is added during the mashing process and is denatured and inactivated during the subsequent wort-boiling stage. In a 13-week study in rats. oryzae Mt. oryzae. One colony was selected and subjected to chemical mutagenesis and screening for high yield of laccase. A transformant producing an adequately high quantity of laccase was selected for use as the laccase production strain A. The laccase gene is stably integrated into the A. Toxicological studies were conducted on the LEC.

15 mg TOS/kg of body weight per day). in chewing gum. The manufacturing procedure comprises a fermentation process. produced by a strain of Humicola insolens The mixed b-glucanase and xylanase preparation under evaluation is produced by fed-batch.35 mg TOS/kg of body weight per day for a 60 kg person) for chewing gum. submerged. The ratio between the NOEL of 1700 mg TOS/kg of body weight per day from the 13-week study of oral toxicity and the cumulative intake deriving from all these conservative estimates is nearly 2000. hemicellulase. giving an estimated daily intake of 9 mg TOS/day (equivalent to 0. Ultrafiltration and/or 28 . The production strain has been selected for improved enzyme production. The cell mass and other solids are separated from the broth by filtration or centrifugation. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established. 3. pentosans and other gums in order to reduce the viscosity of the solution and thereby increase the filtration rate of both wort and beer and improve beer clarity. Moreover. The Committee allocated an ADI “not specified” to laccase from this recombinant strain of A.Nevertheless.g. including cellulase.2 mg TOS/day (equivalent to 0. The preparation is used in beer brewing to hydrolyse b-glucans. and several secondary activities.05 mg TOS/kg of body weight per day for a 60 kg person) for toothpaste. a purification process. pentosanase and arabinase.8 mg TOS/day (equivalent to 0. b-glucanase and xylanase. since the Committee is aware that laccases are receiving increasing interest for various applications other than brewing. used in the applications specified and in accordance with good manufacturing practice. conservative estimates of daily intakes resulting from these uses were performed. This enzyme mixture has not been evaluated previously by the Committee. resulting in the following values: 2 mg TOS/day (equivalent to 0.1. oryzae. breath mints and toothpaste. mouthwash.26 mg TOS/kg of body weight per day for a 60 kg person) for mouthwash. pure culture fermentation of a strain of Humicola insolens that is non-pathogenic and non-toxigenic. 12.21 mg TOS/kg of body weight per day for a 60 kg person) for breath mints and 3. e. a very conservative estimate of daily intake from beer was performed with the assumption that all the TOS would persist in the final product. 16 mg TOS/day (equivalent to 0. b-glucanase enzyme preparation.7 Mixed xylanase. a formulation process and finally quality control of the finished product. The enzyme preparation contains two main activities.

In conclusion.1 mg TOS/day (equivalent to 0. giving an estimated daily intake of 5. very conservative estimates of daily intakes were performed with the assumption that all the TOS would persist in final products. The enzyme preparation is added during the mashing process of beermaking and the enzymes are denatured and inactivated during the subsequent wort-boiling stage. Compared to the NOEL of 0. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro. The Committee is not aware of any other uses for this enzyme mixture in which the enzymes might persist in the final product. the margin of safety is nearly 5000. used in the applications specified and in accordance with good manufacturing practice. no residual enzymes are expected to be present in food processed using this enzyme preparation. The preparation may also be used in the spirits industry. Therefore this highest dose (equivalent to 0.085 mg TOS/kg of body weight per day) for beer and 2. Nevertheless. The materials added to the LEC upon stabilization and formulation/standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns.evaporation are used for concentration and further purification. in this case no enzymes or other organic solids are expected to be carried over into the final product because ethanol is removed by distillation from the fermentation mash containing the enzyme preparation. Toxicological studies were conducted on the LEC. In a 13-week study in rats. The beer filtration process is likely to remove the denatured enzymes along with other insoluble materials. and potassium sorbate.2 g LEC/kg body weight per day. again.62 g total organic solids (TOS)/kg of body weight per day) is considered to be the NOEL.04 mg/kg of body weight per day for a 60 kg person) for spirits. glycerol.62 g TOS/kg of body weight per day from the 13-week study of oral toxicity. The liquid enzyme concentrate (LEC) is then stabilized and formulated/ standardized by the addition of sorbitol. 29 . no significant treatment-related effects were seen when LEC was administered by oral gavage at doses of up to and including 10.7 mg TOS/day (equivalent to 0. insolens. The Committee allocated an ADI “not specified” to mixed bglucanase/xylanase from the production strain H.

depending on the food matrix in which it is used. Pharmacokinetic analysis of plasma metabolites after oral administration of radiolabelled neotame to rats indicated that peak plasma concentrations occurred after 0.3-dimethylbutyraldehyde in a one-step chemical synthesis.8 Neotame Neotame is a dipeptide methyl ester that is intended for use in food as a sweetener and flavour enhancer in a variety of applications. followed by purification. the common name for N-[N-(3. kidney. The metabolism and pharmacokinetics of neotame have been examined in mice. is manufactured from aspartame and 3.6 hours). followed by a rapid decline with a t1/2 of approximately 1 hour. while other metabolites each accounted for <5%. and milling. rats.3. Neotame.4 hours followed by rapid clearance (t.1. a white/off-white powder.3-dimethylbutyl)-La-aspartyl]-L-phenylalanine and methanol. Approximately 20–30% of orally-administered neotame is absorbed in all species studied. 90– 95% of the radioactivity was recovered in the urine and faeces within 48 hours.3-dimethylbutyl)-L-aaspartyl]-L-phenylalanine 1-methyl ester.5 hours. It has a sweetness potency of 7000–13 000 times that of sucrose and 30–60 times that of aspartame. Neotame. More than 95% of orallyadministered neotame is metabolized. radioactivity was not detected in the fetus. Neotame has not been evaluated previously by the Committee. In a study undertaken in pregnant rats. with a maximum plasma concentration at 0. Following oral administration of radiolabelled neotame to rats and dogs. The major metabolite found in the urine and faeces of the rat and dog was de-esterified neotame. is chemically related to aspartame. with lower concentrations of radioactivity detected throughout the rest of the body. In studies in rats given radiolabelled neotame by gavage. Unchanged neotame was not detected in rat urine but was present at 1–6% of the administered dose in the urine of dogs. radioactivity was found to be primarily confined to the stomach. which includes a reductive alkylation. The major metabolic pathway for both absorbed and nonabsorbed neotame is de-esterification to N-[N-(3. rabbits and humans. dogs. Concentrations of de30 . a reaction which is mediated by non-specific esterases. Unchanged neotame was not detected in the faeces of rats or dogs. De-esterified neotame accounted for approximately 80% of the neotame administered. liver. and bladder. gastrointestinal tract. 0. There was no evidence of accumulation of radioactivity in any tissue. Pharmacokinetic analysis of absorbed neotame in human plasma after oral administration of radiolabelled neotame indicated rapid absorption. drying.

98% of the radioactivity was recovered in the urine and faeces within 72 hours. Short-term and long-term studies on neotame have been conducted in mice. The Committee considered that this effect was due to reduced palatability of the neotame-containing diet rather than neotame-induced toxicity. the high incidence of food scattering is indicative of reduced palatability of the diet. with no evidence of tissue accumulation. in the 13-week study in mice fed neotame in the diet. as would be expected if the observed changes were a manifestation of treatment-related toxicity. Fourth. there were no changes in body-weight gain or food conversion efficiency in rapidly growing rats consuming neotame-containing diets. the Committee considered that neotame is rapidly but only partially absorbed in all species studied. which was linked in most cases to a measurable decrease in food consumption. There was no significant decrease in body 31 . First. The unchanged neotame detected in the urine represented 3. On the basis of the data evaluated. After oral administration of radiolabelled neotame to humans. is itself eliminated rapidly via the urine and faeces. when the effect of neotame on palatability of the diet was specifically examined in a preference study comparing basal diets with and without neotame at concentrations of 50–15 000 mg/kg diet. in the 1-year rat study. Second.3% of the administered dose.4% of the administered dose of neotame. while no unchanged neotame was found in the faeces. A metabolite detected initially only in human urine (and subsequently also in female rat urine) was identified as 3. and that both absorbed and non-absorbed neotame are metabolized via well-characterized pathways to non-toxic metabolites. reduced food consumption often occurred at the start of treatment at all doses. the body-weight changes observed in the rat and dog 13-week studies were partially reversible when the animals were returned to a basal diet during the 4-week period at the end of the study. De-esterified neotame was the major metabolite in both urine and faeces. Third. In all of these studies. followed by some degree of adaptation as the animals adjusted to the diet. Fifth. rats and dogs using a body-weight adjusted constant dose regime. de-esterified neotame. Sixth.esterified neotame in plasma peaked at 1 hour and declined with a t1/2 of 1.3-dimethyl-butanoyl-L-carnitine and represented 0. The major metabolite.5 hours. the rats showed a clear preference for the diet without neotame when the neotame concentration was ≥150 mg/kg diet. This conclusion is supported by several observations. studies were conducted over a wide range of doses (50–1000 mg/kg of body weight per day) and body-weight changes were not closely related to dose.5–3. the major effect observed was a treatment-related decrease in body-weight gain. particularly at high doses.

when the reduced palatability significantly decreased food consumption. On the basis of these changes. there were changes in clinical chemistry parameters in dogs fed ≥600 mg neotame/kg of body weight per day.weight observed in this study. there was no treatmentrelated increase in tumour incidence at doses of up to 4000 mg neotame/kg of body weight per day. the NOEL was 1000 mg neotame/kg of body weight per day in the diet. In the 13-week study in mice. with no clinical signs of toxicity. there was a small but significant increase in serum alkaline phosphatase activity at 1000 and 3000 mg neotame/kg of body weight per day in the diet at week 13. Aside from the palatability-related decreases in body-weight gain. In the light of the above information. Nevertheless. no increases in alkaline phosphatase activity were observed and the NOEL. on the basis of an increase in liver weight relative to body weight. the Committee considered this to be indicative of a treatment-related effect and it was therefore used as the basis of the NOEL of 200 mg neotame/kg of body weight per day. was 1000 mg neotame/kg of body weight per day. in the 1-year study in rats following exposure in utero. neotame was well tolerated in all species at high doses in the diet in both the short-term and long-term studies. the NOEL was 300 mg neotame/kg of body weight per day. the only significant change was an increase in alkaline phosphatase activity (hepatic isoenzyme) that was observed only at the high dietary dose (800 mg/kg of body weight per day) in both males and females. the Committee agreed that the NOELs for the various short-term and long-term studies of toxicity should not be assigned on the basis of decreases in body weight or body-weight gain. In the 2-year study of carcinogenicity in mice. there was no treatment-related increase in tumour incidence at doses of up to 1000 mg neotame/kg of body weight per day. The increase in alkaline phosphatase activity was rapidly reversible and was not accompanied by changes in other parameters indicative of cholestasis or other hepatotoxicity. including significant increases in serum alkaline phosphatase activity. 32 . However. The small increase in alkaline phosphatase activity observed at 200 mg/kg of body weight per day in 3 out of 4 female dogs was not considered to be toxicologically significant and a NOEL of 200 mg neotame/kg of body weight per day was established. based on the highest dose tested. In the 2-year study of carcinogenicity undertaken in rats following exposure in utero. but body-weight gain was reduced in males at 5000 mg/kg diet and above. which isoenzyme analysis confirmed to be of hepatic origin. In the 13-week study in dogs. In the 13-week study in rats. In the 1-year study in dogs.

the Committee considered this to be related to a decrease in food consumption as a result of the reduced palatability of the diet containing neotame. The major effect observed was a reduction in body-weight gain in treated animals compared to controls. on the parameters associated with the cardiovascular. parturition. on the basis of the highest dose tested. Studies of genotoxicity have examined the ability of neotame to induce gene mutations in both bacterial and mammalian cells. In rats. fertility. In rabbits. respiratory or renal systems in dogs. was 1000 mg neotame/kg of body weight per day.and long-term studies. as well as chromosome aberrations in vitro in Chinese hamster ovary cells and in vivo in mouse bone marrow cells. body weight or body-weight gain. offspring viability. The Committee considered that the NOEL in this study. The developmental toxicity of neotame was examined in rats and rabbits at doses of up to 1000 mg neotame/kg of body weight per day and 500 mg neotame/kg of body weight per day. compared to controls during the pre-weaning days 14–21 when pups begin to consume solid food. there was no significant effect on body weight or bodyweight gain during gestation. As in the short. probably as a result of a decrease in food consumption. and on the hexobarbital-induced sleeping time in rats were examined. The potential pharmacological effects of neotame on the gastrointestinal system in rats. There was no evidence of genotoxicity in any of the tests. In neither species was there any evidence of embryotoxicity or teratogenicity. there were no treatmentrelated effects on reproductive parameters (estrus cycle. Mean litter body weights in treated groups in both the F1 and F2 generations were also reduced at day 21. on the autonomic nervous system in guinea pig ileum. however. gestation time. mating performance. respectively. sex ratio. there was no significant effect on overall food consumption. These studies demonstrated no evidence of pharmacological activity associated with neotame. Studies of toleration in humans included a single-dose study in adult males. and gestation index) or on litter size.In a study of reproductive toxicity in rats. physical development or learning at doses of up to 1000 mg neotame/kg of body weight per day. The significant increase in the swim time for F1 males in a waterfilled Y-maze at 1000 mg/kg of body weight per day was within the variability seen in studies of this type and was likely to be related to reduced body weight rather than reduced learning ability. 2-week studies in both diabetic and non-diabetic male and 33 . there was an immediate but transitory decrease in food consumption resulting in lower body-weight gain in the treated animals.

of high statistical significance and treatment-related. Although a 1-year study is not considered to be a long-term study in dogs. and a 3-month study in male and female adults. While the increase in alkaline phosphatase was moderate. In both the 2-week studies on diabetic and non-diabetic adults. The Committee agreed there were insufficient data to discount this effect and therefore accepted the dog as the most sensitive species with a NOEL for neotame of 200 mg/kg of body weight per day. and was not accompanied by other evidence of liver toxicity. Furthermore. there were no signs or symptoms associated with the administration of neotame at doses of up to 1. Single doses of 0. an additional safety factor was not considered necessary. mutagenic. On the basis of the available studies.5 mg neotame/kg of body weight per day were tolerated without treatment-related signs or symptoms. Studies of toleration in humans confirmed the lack of any treatment-related signs or symptoms at doses of up to 1. reversible. neotame was well tolerated at doses of up to 1. no treatment-related adverse effects were observed in a 4-week study in rats fed with a mixture of the three minor degradation products. there were no signs or symptoms associated with the administration of neotame.5 mg/kg of body weight per day.5 mg neotame/kg of body weight per day in diabetic and nondiabetic subjects. including humans. the observed change was reproducible. treatment with neotame had no effect on plasma glucose or insulin concentrations. Evaluation. 34 .5 mg/kg of body weight per day. Appropriate studies indicated that neotame is not carcinogenic. which accounted for 7% of the initial neotame concentration after 8 weeks storage. In the study in diabetic adults. The only consistent treatment-related effect observed was an increase in serum alkaline phosphatase activity in the 13-week and 1-year studies in dogs fed neotame in the diet. In the 3-month study of toleration in non-diabetic adults. on the basis of the 1-year study in dogs fed neotame in the diet.female adults. All of the degradation products have low acute toxicity and gave negative results in tests for genotoxicity. in light of the human data. The major degradation product of neotame under normal storage conditions was de-esterified neotame. teratogenic or associated with any reproductive/developmental toxicity. The Committee also noted that the safety studies conducted in animals and humans with neotame would have included low levels of these degradation products. the Committee considered neotame to be a substance of low toxicity across a range of species. Three minor degradation products were formed which represented <1% of the initial neotame concentration. In both of these 2-week studies.

35 . the results were consistent with very low toxicity and showed no evidence for carcinogenicity. Neotame is intended for use as a tabletop sweetener as well as in a large variety of solid and liquid foods. sealing and surface finishing agent in food products such as dairy-based desserts. Conservative calculations based on its lowest sweetness potency (7000 times that of sugar) suggest that intakes of 2 mg neotame/kg of body weight per day would correspond to the replacement of 840 g of sugar in the diet of a 60 kg adult. followed by partial hydrolysis of the resulting ester in the presence of an alkaline catalyst. the Committee was able to conclude that polyvinyl alcohol was very poorly absorbed after oral administration.The Committee established an ADI of 0–2 mg/kg of body weight for neotame on the basis of a NOEL of 200 mg/kg of body weight per day in a 1-year study in dogs and a 100-fold safety factor. The number of acetate groups in polyvinyl alcohol is determined by the degree of hydrolysis (86. or from studies that were not conducted in accordance with GLP principles or that were conducted with material that did not comply with the specification for polyvinyl alcohol as prepared at the current meeting.9 Polyvinyl alcohol Polyvinyl alcohol is a synthetic resin that is prepared by polymerization of vinyl acetate. The Committee agreed that the ADI also applied to those individuals with phenylketonuria since the formation of phenylalanine from the normal use of neotame would not be significant in relation to this condition. in the range of 0. Therefore even a total replacement of sugar with neotame would not lead to the ADI being exceeded.8% by weight.5–89.1. Assessment of intake. Polyvinyl alcohol has not been evaluated previously by the Committee. binder.0% hydrolysis for this food additive specification). The Committee examined a large database of studies of the toxicity of polyvinyl alcohol after administration by various routes to a number of species. confectionery and cereal products and dietary supplement tablets. Polyvinyl alcohol is used as a coating. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established. Nonetheless. 3. that the acute oral toxicity was generally very low and that. not relevant to oral administration. taken as a whole.2– 1. Much of the information was found to be dated.

blood coagulation. The Committee considered that these observations did not represent adverse effects. which had been performed with preparations of polyvinyl alcohol complying with the food additive specification. clinical chemistry. In both the 90-day and the two-generation studies. Evaluation. that provided no evidence for local or systemic carcinogenic activity. A 90-day study of toxicity in rats treated orally revealed no toxicity at doses of up to 5000 mg of polyvinyl alcohol/kg of body weight per day. on the basis of the maximum dose tested from both the 90-day and the two-generation studies in rats. urinalysis. that was considered to be directly relevant to the safety evaluation of oral exposure to polyvinyl alcohol. the very poor absorption of preparations of polyvinyl alcohol complying with the specification following oral administration and the absence of any effects on the gastrointestinal tract in the 90-day study in rats. the most notable observations were loose or unformed stools in the groups given higher doses of polyvinyl alcohol. and reviewed a study involving the intravaginal administration of polyvinyl alcohol to mice. No toxicity was observed in a two-generation study of reproduction in rats in which the parental. 5 days per week for 104–105 weeks. The Committee noted the lack of reports of any apparent toxic or carcinogenic effects in studies concerning polyvinyl alcohol as a whole. in any species. organ weight or macroscopic pathology were observed. No significant differences in body weight. including the gastrointestinal tract. This was the only short-term study provided. neurobehaviour. There was no evidence for genotoxicity in a battery of tests undertaken with preparations of polyvinyl alcohol complying with the food additive specification. this being attributed to the high intestinal concentration of unabsorbed test material and increased food consumption in these groups. The Committee also noted a report that more polyvinyl alcohol was absorbed after intravaginal than oral administration. haematology. from the control group and the groups given high doses showed no evidence for treatment-related pathology. first and second generations received a maximum dose of polyvinyl alcohol of 5000 mg/kg of body weight per day. Despite the absence of long-term studies or studies in a second 36 . and which met appropriate GLP standards.The Committee also considered a number of recent studies. Microscopic examination of an extensive range of organs and tissues. The Committee identified a NOEL of 5000 mg/kg of body weight per day for polyvinyl alcohol.

1. 37 . the Committee reviewed new information related to the chemical charaterization of quillaia extracts and further information related to the specifications. the ADI was made temporary pending further clarification. No ADI was allocated to the semi-purified product. Extreme intakes based on Australian and New Zealand consumption data during one day were shown to reach 2–2. with a safety factor of 100.5 g/day at the 97. The Committee emphasized again that the evaluation of (unpurified) quillaia extract at its twenty-sixth and twenty-ninth meetings was limited to the commercial product that had been tested at that time.10 Quillaia extracts Unpurified quillaia extracts were reviewed by the Committee at its twenty-sixth and twenty-ninth meetings (Annex 1.species. reference 154). previously designated as “semi-purified”. 3.5th percentile. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established. An ADI of 0–5 mg/kg of body weight for unpurified quillaia extracts was allocated at the latter meeting. At the fifty-seventh meeting (Annex 1. one for quillaia extract (type 1) previously designated as “unpurified”. the Committee considered the data adequate for the establishment of an ADI. references 59. The intake estimate based on use levels provided by the sponsor and national food consumption data (from the USA) shows a mean ingestion of around 0. the Committee reviewed quillaia extracts and adopted tentative specifications. At its present meeting.5 g/day. to replace the existing specifications. It was agreed that two separate specifications should be developed. Assessment of intake. A chemical and technical assessment (CTA) for products of type 1 and type 2 was prepared. 70). and to a lack of information on the composition of the saponin and non-saponin fractions of the preparations used in this study and in the previous 90-day studies. Owing to outstanding queries concerning the relevance of a 90-day study in rats that had become available to the Committee after the ADI was allocated. corresponding to 33 and 42 mg/kg of body weight per day respectively. on the basis of the NOEL of 5000 mg/kg body weight per day from the 90-day and two-generation studies in rats. the other for quillaia extract (type 2).3 mg/kg of body weight per day for a 60 kg adult. equivalent to 8. as requested at the fifty-fifth and the fifty-seventh meetings. The Committee therefore established an ADI for polyvinyl alcohol of 0–50 mg/kg body weight per day.

such as polyphenols. The authors identified two separate profiles in this random group of isolates. Four saponins (QS-7. ~32% carbohydrates (mainly sugars) and ~5% fat. ~7% tannins. trees were screened in a search for plant varieties that exhibit a specific saponin spectrum. followed by clarification and purification. QS-18. ~14% salts (mainly calcium oxalate). The Committee noted that a considerable amount of data was available that characterizes the composition of the saponin fraction in more detail. as neither soil. with reference to a recent study (3) on the variability of saponins in quillaia extracts. altitude. The authors showed that the two major peaks present in profile A were identical to those of the saponins QS-18 and QS-21. QS-17. saponaria Molina (family Rosaceae). Bark extracts obtained from thirty different natural. Comparison with the saponin profile of a commercial extract revealed that the latter displayed a mixture of both profiles. These saponins are also considered to be representative of the total saponin content and their quantification is henceforth the basis for the assay. The product contains triterpenoid saponins consisting predominantly of glycosides of quillaic acid. carbohydrates and salts. not cultivated. showing two predominant saponin peaks. with a total saponin content of 20–26%. of which one (profile A). Other substances that occur in bark tissue. Profile A and profile B were each observed in 50% of the trees sampled. appeared to be a subset of the other (profile B). The non-saponin fraction was described in the literature as consisting of 10–16% polyphenols. The observed variation in saponin profiles between trees was attributed by the authors to genetic factors. or age of trees or sampled tissues correlated with the composition of saponins. The question of whether the product that had been tested toxicologically was representative of the material currently being marketed was considered by the Committee. tannins. in addition. 38 . it was observed that trees from the same location could display different profiles. As quillaia extract is a product of natural origin. it was recognized that the proportions of these four compounds (and other saponins) may vary.Quillaia extract (type 1) Quillaia extract (type 1) is obtained by aqueous extraction of the milled inner bark or of the wood of pruned stems and branches of Q. The Committee noted that these figures were only approximate and understood that the content of these plant constituents might vary between batches. This mixed profile was attributed to the mixing of barks from both tree types during processing. described 10 years earlier. QS-21) have been identified as the major constituents of that fraction. are also present.

The intake estimates made on the basis of consumption data for soft drinks likely to contain quillaia extract (type 1) were prepared by the Committee at its fifty-seventh meeting and remain valid. The “temporary” assignment for the ADI of 0–5 mg/kg of body weight was removed.to four-fold concentrations of saponins are subsequently achieved by ultra-filtration or affinity chromatography. For a more recently developed category of frozen carbonated beverages. concentrations of up to 250 ppm have been reported. the year when purity criteria for quillaia extract were laid down by the Emulsifiers and Stabilisers in Food Regulations of the United Kingdom. Assessment of intake. saponaria Molina (family Rosaceae) is subjected to several clarification and purification steps. such as lactose. yielding more purified extracts with saponin contents of 75–80%. Three. Quillaia extract (type 2) The initial manufacturing steps of quillaia extract (type 2) are the same as those for type-1-extract: an aqueous extraction of the inner bark or wood of Q. the Committee agreed that the material tested toxicologically was representative of the material specified as quillaia extract (type 1). Most of the products available are diluted with carriers. in order to standardize the saponin concentration and thereby achieve a consistent range of foam-building strength. Evaluation.The Committee also received confirmation from a European manufacturer that no significant changes had been introduced to the manufacturing process since 1975. primarily for its foaming properties. a feature which is assumed not to change significantly within a period of 20 years within a particular population of trees. The material tested before 1982 complied with these specifications. and considering further that mixing of batches with different saponin profiles would take place during processing. The Committee noted that the saponin profile of quillaia extract (type 2) obtained using ultra-filtration was similar to the saponin profile 39 . In view of the new data suggesting that the variability of the saponin content in single trees is determined genetically. maltodextrin or maltitol. New specifications were prepared for quillaia extract (type 1). Quillaia extract (type 1) is used in food applications. The non-saponin fraction of the type-2 extracts contains same minor components as that of the type-1-extracts. Concentrations at which the undiluted extract is used in soft drinks were reported to be approximately 100 ppm (dry basis).

it was also recognized that these four compounds (and other saponins) may be present in varying proportions. since the manufacturer applied a method involving partial hydrolysis of the saponins in the sample.11 Xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum The enzyme preparation under evaluation contains the enzyme xylanase. for example. Concentrations of saponins obtained using this method could not be related to those obtained using the method described in the specifications. In addition. however. marketing data related to the percentage of consumers of the products would be necessary in order to refine the estimate. The Committee concluded that the limited information on the qualitative and quantitative composition of the type-2 extracts prevented the Committee from considering the establishment of an ADI or assessing the need for additional toxicological studies. inserted by recombinant DNA tech40 . Assessment of intake. at higher concentrations. No information on saponin profile was available for the material after affinity chromatography. in order to assess the per capita intake of the additive in different regions. Xylanase is produced by submerged fermentation of a strain of Fusarium venenatum that is non-pathogenic and non-toxigenic (under conditions consistent with good manufacturing practice). 3. Quillaia extract (type 2) is used in Japan as an emulsifier for preparations containing lipophilic colours or flavours that are added to. Limited data were available on the composition of the non-saponin fraction. As quillaia extract is a product of natural origin. The manufacturer claimed that the extract produced by ultra-filtration contained <8% polyphenols and <8% tannins. which has not been evaluated previously by the Committee.(QS-7. <5% salts and <5% sugars.1. and which has been genetically modifed to carry a gene encoding a xylanase from Thermomyces lanuginosus. The typical concentration of type-2 extract in these food products was claimed to be not more than 10 ppm. soft drinks. QS-21) displayed when the extract was tested according to the assay method described in the specifications. New specifications were prepared for quillaia extract (type 2). Evaluation. It was estimated that the extract produced using affinity chromatography contained <10% polyphenols and tannins. QS-18. QS-17. fermented vegetables and dressings. The Committee recommended that production data for quillaia extract (type 2) and consumption data for products containing the additive should be collected.

B strain. The JRoy36-19.B strain was subsequently transfected with DNA containing the amdS gene from Aspergillus nidulans. the major trichothecene produced by non-engineered strains of F. venenatum strain used in the production of mycoprotein marketed for human consumption since 1985 under the trade name “Quorn. the concentrations of these secondary metabolites. the transformed strain was evaluated for production of diacetoxyscirpenol (DAS). One of the transformants was designated as LyMC4. venenatum: no DAS was detected. is a spontaneous mutant of the F. in turn.B produces these secondary metabolites to the same extent under industrial fermentation conditions. venenatum is capable of producing other secondary metabolites. The amdS gene was flanked by specific sequences of the F. are considered to be of no toxicological relevance. venenatum under conditions known to be optimal for production of these secondary metabolites revealed them to be present only at very low concentrations. Deletion of the tri5 gene served to inactivate the biochemical pathway by which mycotoxins (trichothecenes) are synthesized. The MLY3 strain is a spontaneous mutant of the CC1-3 strain that. such as culmorins. The selected strain was designated as JRoy36-19.B. which hydrolyses xylosidic linkages in the arabinoxylans into smaller oligosaccharides. 41 . if present. The enzyme preparation is used in baking applications to increase the elasticity of the gluten network. dextrin. improving handling and stability of the dough. resulting in a liquid enzyme concentrate (LEC). sorbitol. The enzyme is subsequently partially purified and concentrated. venenatum host strain MLY3. A single transformed colony producing xylanase was selected.B and was used as a xylanase production strain. lanuginosus and the bar gene from Streptomyces hygroscopicus. The enzyme produced is an endo-xylanase. and wheat solids. The enzyme is denatured and inactivated during bread baking. In the final preparation. enniatins and fusarins.” The MLY3 strain was transfected with the expression plasmid pjRoy36 containing the xylanase gene from T. To confirm that the tri5 gene had been deleted.B was developed from the F.niques. The production strain LyMC4. venenatum tri5 gene and replaced the tri5 gene in the JRoy36-19. this LEC is stabilized and standardized/formulated with sodium chloride. The bar gene confers resistance to the herbicide phosphinothricin and serves as a selectable marker. Analyses performed on conventional F. F. As it is very unlikely that the production strain LyMC4.

no significant treatment-related effects were seen when LEC was administered by oral gavage at doses of up to and including 10 ml LEC/kg of body weight per day.2 Magnesium silicate (synthetic) This additive was on the agenda of the sixty-first meeting in response to a request by the Codex Committee on Food Additives and 42 . Compared to the NOEL of 1. the margin of safety is nearly 10 000. and formulated a general method of analysis for residual solvents. 3.12 mg TOS/kg of body weight per day). The materials added to the LEC for stabilization and formulation/standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns. The Committee allocated an ADI “not specified” to xylanase from this recombinant strain of F. using head-space gas chromatography with flame ionization detection (FID). The existing tentative specifications were revised to refer to this general method and the “tentative” designation was removed. In a 13-week study in rats.1 g total organic solids (TOS)/kg of body weight per day) was considered to be the NOEL.2. the Committee considered the methods supplied.1 g TOS/kg of body weight per day derived from the 13-week study of oral toxicity.2. the specifications for b-carotene from Blakeslea trispora were made tentative and the Committee requested additional information regarding the analysis of residual solvents. venenatum.The xylanase was assessed for potential allergenicity by amino acid sequence comparison with known allergens listed in publiclyavailable protein databases. The LEC was not mutagenic in an assay for mutagenicity in bacteria in vitro and not clastogenic in an assay for chromosomal aberrations in mammalian cells in vitro.9 mg TOS/day (equivalent to 0. A toxicological monograph and a chemical and technical assessment (CTA) were prepared and specifications were established. At the present meeting.2 Revision of specifications At the fifty-seventh meeting. used in the applications specified and in accordance with good manufacturing practice. 3. Conservative estimates of daily intakes resulting from the use of xylanase in bakery goods are 6.1 b-Carotene from Blakeslea trispora 3. Toxicological studies were conducted on the LEC. No immunologically-significant sequence homology was detected. Therefore this highest dose tested (equivalent to 1.

The evidence provided showed that magnesium silicate removes free fatty acids and other polar compounds from used cooking oils. whereas industrial fryers use £2% by weight. Regular filtration through magnesium silicate can extend the usage time of a frying oil by ≥50%. The Committee decided that the tentative specifications for the two additives would be withdrawn if the requested information was not received before the end of the year 2004. A chemical and technical assessment (CTA) was prepared. reference 154). pending information on levels and the method for loss on drying for the monohydrate. new information on the method of manufacture was provided and was also incorporated into the revised specifications. For trisodium diphosphate. and on the assay method for the hydrates.Contaminants at its Thirty-fourth Session (4) that the Expert Committee examine evidence for its functional use as a filtering aid and adsorbent. information on limits and method for loss on ignition for the monohydrate and on loss on drying for the anhydrous material was received. depending on the oil used and the type and amount of food fried. polar compounds (such as fatty acids) are adsorbed and held for removal by filtration. A chemical and technical assessment (CTA) for both phosphates was prepared. The tentative status of the specifications was maintained. For monomagnesium phosphate.3 Monomagnesium phosphate and trisodium diphosphate At its fifty-seventh meeting (Annex 1. the Committee designated the specifications for monomagnesium phosphate and trisodium diphosphate as tentative and information was requested on limits and methods for the loss on drying and loss on ignition. If magnesium silicate is mixed into a used frying oil. 3.2. no information was received. Restaurants typically use £1% by weight of this filtering aid. The magnesium silicate is discarded after it has been used to remove impurities. The information on the method for loss on drying for the hydrates is necessary in order to express the assay for these additives in terms of dry weight. 43 . The specifications were revised and the Committee maintained the tentative status.

After considering the information provided. or by extraction from sucroglycerides. reference 154). The substance was placed on the agenda in response to a request by the Codex Committee on Food Additives and Contaminants at its Thirty-fourth Session that the specifications should be revised. the Committee revised the method of analysis for the determination of acid-soluble substances to include temperature conditions for the digestion of the sample. Methods of analysis for the determination of residual levels of solvents (dimethylformamide. di. 3.5. 44 . the methods specify the use of packed gas chromatography columns and instruments that are now considered to be obsolete (see general consideration 2. ethyl acetate and methyl ethyl ketone) used in the manufacture of the substance were revised to include more modern gas chromatographic conditions than those included in the current specifications.2.2.6 Talc Talc was placed on the agenda of the present meeting following a request for the revision of the specifications. by removing the step using dimethylformamide in favour of a method using a suspension in water. i.e. A new provision and method of analysis for amphiboles and serpentines for the detection of asbestos was also included in the specifications. lead content and the method of assay. After receipt of information regarding the measurement of pH. They are prepared from sucrose and methyl or ethyl esters of food fatty acids by esterification in the presence of a catalyst. specific rotation. the Committee decided to modify the method by which pH is measured. isopropanol. isobutanol. including the use of capillary columns. 3. For example. The Committee removed the “tentative” designation from the specifications.2). loss on drying.2. methanol.5 Sucrose esters of fatty acids Sucrose esters of fatty acids consist of mono-. The revised methods submitted to the Committee have been thoroughly validated and the Committee found them suitable for their intended purposes. The existing specifications were revised.and tri-esters of sucrose with fatty acids commonly occurring in food. Methods of analysis for the determination of free sucrose and solvent residues in addition to those noted above (dimethyl sulfoxide and propylene glycol) that are currently included in the specifications may need revision to bring them up to date.3.4 Natamycin The tentative specifications for natamycin were revised in response to the queries raised by the Committee at its fifty-seventh meeting (Annex 1.

143. Eighty-eight substances remain to be reviewed at future meetings. the chemical is first assigned to a structural class as identified by the Committee at its forty-sixth meeting (Annex 1. • Class II.3 Revision of limits for metals in food additives The Committee reviewed the limits for heavy metals in the specifications for a group of food additives. Where no data were submitted. Substances in this class may contain reactive functional groups.3. 137. reference 122). the Committee reviewed limits for heavy metals in 39 food additives. the Committee followed the guidelines established at previous meetings.1 Flavouring agents Flavouring agents evaluated by the Procedure for the Safety Evaluation of Flavouring Agents Five groups of flavouring agents and additional compounds for two previously published groups were evaluated using the Procedure for the Safety Evaluation of Flavouring Agents as outlined in Figure 1 (Annex 1. 4. • Class III. the Committee used the following definitions. colour retention. 4. For the purpose of the evaluations. reference 149). adapted from the report of its forty-sixth meeting: 45 . A key element of the Procedure involves determining whether a flavouring agent and the product(s) of its metabolism are innocuous and/or endogenous substances. 154 and 160). carrier solvent. The structural classes are as follows: • Class I. In applying the Procedure. references 116. Flavouring agents that have structural features that permit no strong initial presumption of safety. and sequestrant applications. 131. 122. or may even suggest significant toxicity. clouding agent. The functional uses of the additives reviewed included absorbent. Flavouring agents that have simple chemical structures and efficient modes of metabolism which would suggest a low order of toxicity by the oral route. The outdated tests for heavy metals (as lead) in these food additive specifications were replaced with appropriate limits for individual metals of concern (see Table 1). antioxidant. At its present meeting. antifoaming. continuing the 5-year programme initiated at the fifty-fifth meeting (Annex 1. 149. Flavouring agents that have structural features that are less innocuous than those of substances in Class I but are not suggestive of toxicity. emulsifier.

dl-a Triethyl citrate — 522 520 300 304 (i) 304 (ii) 542 320 321 302 385 519 389 386 312 315 — 585 384 322 311 900 1521 326 452 (ii) 310 541 (i) 301 — 316 325 512 444 319 388 307 b 307 a 307 c 1505 3 — — — — — 3 — — — — — — — — — — — — — — — — — 3 — 3 — — — — — — — — — — — — Limits (mg/kg) Lead 5 5 5 2 2 2 2 2 2 2 2 10 2 2 2 2 2 1 2 2 2 1 1 2 4 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Cadmium — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — Mercury — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — INS: International Numbering System. —: not applicable. mixed Tocopherol concentrate. acidic Sodium ascorbate Sodium caseinate Sodium erythorbate Sodium lactate (solution) Stannous chloride Sucrose acetate isobutyrate Tertiary butylhydroquinone Thiodipropionic acid Tocopherol concentrate. Arsenic Activated carbon Aluminium potassium sulfate Aluminium sulfate (anhydrous) Ascorbic acid Ascorbyl palmitate Ascorbyl stearate Bone phosphate Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) Calcium ascorbate Calcium disodium ethylenediaminetetraacetate Cupric sulfate Dilauryl thiodipropionate Disodium ethylenediaminetetraacetate Dodecyl gallate Erythorbic acid Ethyl protocatechuate Ferrous lactate Isopropyl citrate mixture Lecithin Octyl gallate Polydimethyl siloxane Polyethylene glycols Potassium lactate solution Potassium polyphosphates Propyl gallate Sodium aluminium phosphate.Table 1 Limits for metals in food additives Additive name INS No. 46 . mixed Tocopherol.

Determine structural class 2. or does a NOEL exist for structurally related substances which is high enough to accommodate any perceived toxicity between the substance and the related substances? Data must be available on the substance or a closely related substance in order to perform a safety evaluation Yes No Yes A5. Do the conditions of use result in an intake greater than 1. Does a NOEL exist for the substance which provides an adequate margin of safety under conditions of intended use.Figure 1 Procedure for the Safety Evaluation of Flavouring Agents 1. Is the substance or are its metabolites endogenous? No A3. Do the conditions of use result in an intake greater than the threshold of concern for the structural class? No No Yes A4. or does a NOEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances? No Additional data required B5. Can the substance be predicted to be metabolized to innocuous products? Yes No A B B3. Does a NOEL exist for the substance which provides an adequate margin of safety under conditions of intended use.5 mg/day? Yes No Substance would not be expected to be of safety concern 47 Substance would not be expected to be of safety concern . Do the conditions of use result in an intake greater than the threshold of concern for the structural class? Yes Substance would not be expected to be of safety concern Yes B4.

48 . an endogenous substance should be judged not to give rise to perturbations outside the physiological range. Manufacturers were requested to exclude use of flavouring agents in pharmaceutical. hormones and other substances with biochemical or physiological regulatory functions are not included. Intake (mg per person per day) = annual volume of production (kg ) ¥ 10 9 (mg kg ) population of consumers ¥ 0. it was assumed that only 60% of the total amount used is reported in Europe and 80% of the amount used is reported in the USA and that the total amount used in food is consumed by only 10% of the population. tobacco or cosmetic products.6 or 0. whether free or conjugated. a survey was conducted in 1995 by the International Organization of the Flavour Industry. Endogenous substances are intermediary metabolites normally present in human tissues and fluids. Intake data Estimates of the intake of flavouring agents by populations typically involve the acquisition of data on the amounts used in food. a series of surveys was conducted between 1970 and 1987 by the National Academy of Sciences National Research Council (under contract to the Food and Drug Administration) in which information was obtained from ingredient manufacturers and food processors on the amount of each substance destined for addition to the food supply and on the usual and maximal levels at which each substance was added in a number of broad food categories. These data were derived from surveys in Europe and the USA.Innocuous metabolic products are defined as products that are known or readily predicted to be harmless to humans at the estimated intake of the flavouring agent. In the USA. In using the data from these surveys to estimate intakes of flavouring agents. or is metabolized to. In Europe. in which flavour manufacturers reported the total amount of each flavouring agent incorporated into food sold in the European Union during the previous year. The estimated intake of a flavouring agent that is.8 ¥ 365 days The population of consumers was assumed to be 32 ¥ 106 in Europe and 26 ¥ 106 in the USA.

Estimated daily per capita intake The total annual volume of production of the 16 flavouring agents in this group is approximately 12 200 kg in Europe and 10 000 kg in the 1 A group of 35 aliphatic lactones of similar structure was evaluated at the forty-ninth meeting of the Committee (Annex 1. black and green tea. reference 132). and the standard formula was applied. alicyclic-fused and aromatic-fused ring lactones The Committee evaluated a group of 16 flavouring agents (see Table 2) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. The Committee had previously evaluated one member of the group. celery stalks. a 14-week study in rats in which the dosage was uncertain owing to loss of the test substance during storage.1. at its thirty-fifth meeting (Annex 1. and — a group of six g.or d-lactones fused to an alicyclic ring (cyclohexyl or decalin ring systems) (Nos 1161–1166). and peppermint oil. No adverse effects were reported. a 90-day study in rats treated with a single dose. At the thirty-fifth meeting. This group included: — a group of four aliphatic lactones (Nos 1157–1160) that form openchain acyclic hydroxycarboxylic acids upon hydrolysis1. soya bean. but the Committee considered these data to be inadequate. The toxicological data considered were derived from studies of acute toxicity in mice. 1171). and guinea pigs. reference 88). but no ADI was established.Several of the flavouring agents that were evaluated at the present meeting were not included in the above surveys or were placed on the market after the surveys were conducted. 4. 1168–1171) have been reported to occur naturally in foods and have been detected in rose-apple. — a group of six g. 1162–1164. 49 .1 Alicyclic. Intakes of these flavouring agents were estimated on the basis of anticipated use by the manufacturer in the USA. 1). the Committee stated that the results of a short-term study in a rodent species and metabolic studies to determine the extent of conversion of dihydrocoumarin to coumarin would be needed before dihydrocoumarin could be re-evaluated. and a study in which three dogs were treated with one of two doses for 2 years (without a control group).or d-lactones fused to a benzene ring (Nos 1167– 1172). dihydrocoumarin (No. Eight of the 16 flavouring agents in this group (Nos 1157. rats. The Committee noted that metabolites of dihydrocoumarin have been identified in rabbit urine.

50

Table 2 Summary of results of the safety evaluations of alicyclic, alicyclic-fused and aromatic-fused ring lactones used as flavouring agentsa No. CAS No. and structure Comments based on predicted metabolism

Flavouring agent

Step A3 Does intake exceed the threshold for human intake?b Step A4 Is the flavouring agent or are its metabolites endogenous?
NR See note 1 No Europe: ND USA: 3

Conclusion based on current intake

Structural class I 4-Hydroxy-4-methyl-5hexenoic acid g-lactone 1157 1073-11-6
O O

No safety concern

(+/-) 3-Methyl-g-decalactone 1158 67663-01-8
O O

No Europe: ND USA: 5 NR No Europe: ND USA: 13 NR

See note 1

No safety concern

4-Hydroxy-4-methyl-7-cisdecenoic acid g-lactone 1159 70851-61-5
O O

See note 1

No safety concern

Tuberose lactone 1160 153175-57-6

No Europe: ND USA: 11

NR

See note 1

No safety concern

O

O

Structural class III Dihydromintlactone 1161 92015-65-1 NR See note 2
O O

No Europe: ND USA: 12

No safety concern

Mintlactone 1162 13341-72-5 NR
O O

No Europe: 4 USA: 9

See note 2

No safety concern

Dehydromenthofurolactone 1163 75640-26-5 NR
O O

No Europe: 2 USA: 9

See note 3

No safety concern

1164 15356-74-8 NR
O O

(+/-)-(2,6,6-Trimethyl-2hydroxycyclohexylidene) acetic acid g-lactone No Europe: ND USA: 0.9

See note 2

No safety concern

Sclareolide 1165 564-20-5
O O

No Europe: 1 USA: 6

NR

See note 2

No safety concern

51

Table 2 (continued) No. CAS No. and structure Comments based on predicted metabolism See note 4

52

Flavouring agent

Step A3 Does intake exceed the threshold for human intake?b Step A4 Is the flavouring agent or are its metabolites endogenous?
NR No Europe: ND USA: 0.07

Conclusion based on current intake No safety concern

Octahydrocoumarin 1166 4430-31-3

O O

1167 65817-24-5
O O

Structural class III 2-(4-Methyl-2hydroxyphenyl)propionic acid g-lactone No Europe: ND USA: 2

See note 5

No safety concern

3-Propylidenephthalide 1168 17369-59-4

No Europe: 20 USA: 52
O

See note 6

No safety concern

O

Yes. The NOELs of 5.42 (males) and 6.55 (females) mg/kg of body weight per day for the related substance 3-propylidenephthalide (No. 1168) are >100 000 times the estimated intake of 2-(4-methyl-2hydroxyphenyl)propionic acid g-lactone in the USA (0.03 mg of body weight per day) when used as a flavouring agent Yes. The NOELs of 5.42 (males) and 6.55 (females) mg/kg of body weight per day are >1000 times the estimated intakes of 3-

55 (females) mg/kg of body weight per day for the related substance 3propylidenephthalide (No.9 mg/kg of body weight per day) when used as a flavouring agent Yes.42 (males) and 6.55 (females) mg/kg of body weight per day for the related substance 3propylidenephthalide (No.3-n-Butylphthalide 1169 6066-49-5 O O No Europe: 0. 1168) is >100 000 times the estimated intakes of 3-n-butylphthalide in Europe (0.42 (males) and 6.6 USA: 0.1 mg/kg of See note 6 No safety concern 53 . The NOELs of 5.2 mg/kg of body weight per day) and in the USA (0. 1168) are >10 000 times the estimated intakes of 3-butylidenephthalide in Europe (0.3 mg/kg of body weight per day) and in the USA (0. The NOELs of 5.01 mg/kg of body weight per day) when used as a flavouring agent Yes.4 See note 6 No safety concern 3-Butylidenephthalide 1170 551-08-6 No Europe: 10 USA: 7 O O propylidenephthalide in Europe (0.

a Step 2: Ten flavouring agents (Nos 1157–1166) in this group are expected to be metabolized to innocuous products. CAS No. NR: not required for evaluation because consumption of the substance was determined to be of no safety concern at step A3 of the decision-tree. The evaluation of these flavouring agents therefore proceeded via the A-side of the decision tree. The aromatic-fused ring lactones (Nos 1167–1172) are in structural class III. . limited metabolic data exists for this subgroup of flavouring agents. Several safety studies are available as discussed in the text See note 8 No safety concern CAS: Chemical Abstract Service. and structure Comments based on predicted metabolism Flavouring agent Step A3 Does intake exceed the threshold for human intake?b Step A4 Is the flavouring agent or are its metabolites endogenous? Conclusion based on current intake Dihydrocoumarin 1171 119-84-6 Yes Europe: 1415 USA: 1111 body weight per day) when used as a flavouring agent Yes. ND: no data on intake reported. The evaluation of these six flavouring agents therefore proceeded via the B-side of the decision tree. Several safety studies are available as discussed in the text See note 7 No safety concern O O 6-Methylcoumarin 1172 92-48-8 O O Yes Europe: 298 USA: 96 Yes.54 Table 2 (continued) No.

4 Hydrolysed to a hydroxycarboxylic acid. 3 Hydrolysed to a ketoacid. respectively.b The thresholds for human intake for structural classes I and III are 1800 and 90 mg/day. followed by excretion as the glucuronic acid or glycine conjugate. followed by excretion as the glucuronic acid conjugate or oxidation of ring substituents to yield polar hydroxylated metabolites that may be excreted in the urine. Notes to Table 2: 1 Hydrolysed to a hydroxycarboxylic acid. The combined per capita intake of flavouring agents in structural class I is 32 mg per day in the USA. followed by b-oxidative cleavage to yield metabolites that are completely metabolized in the citric acid cycle. All intake values are expressed in mg/day. 55 . Excretion of these conjugates either in the free form or as glycine conjugates. 5 Hydrolysed to hydroxycarboxylic acid. followed by glucuronic acid or glycine conjugation. followed by b-oxidative cleavage to a polar hydroxyacid that may be excreted free or conjugated with glucuronic acid. followed by excretion. oxidation of ring substituents or the ring itself to yield polar hydroxylated metabolites that may be excreted in the urine. 6 Hydrolysed to hydroxycarboxylic acid or keto carboxylic acid. 8 Oxidized to yield 7-hydroxy-6-methylcoumarin via ring hydroxylation or coumarin-6-carboxylic acid via methyl group oxidation. The combined per capita intake of flavouring agents in structural class III is 1751 mg per day in Europe and 1305 mg per day in the USA. 7 Hydrolysed followed by b-oxidation to yield o-hydroxybenzoic acid which is excreted primarily in the urine unchanged or as the glycine conjugate. 2 Hydrolysed to a hydroxycarboxylic acid.

metabolism and elimination Lactones are formed by acid-catalysed intramolecular cyclization of 4. most values being at the lower end of this range. Absorption.07–298 mg. lactones would hydrolyse rapidly to the open-chain hydroxycarboxylic acid.USA. distribution. More than 80% of the total annual volume of production in Europe and more than 84% in the USA is accounted for by dihydrocoumarin (No. Metabolic pathways available to aromatic fused-ring lactones (Nos 1167–1172) include excretion as the glycine or glutamine conjugates of the open-chain hydroxycarboxylic acid derivative. 1171). Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1.1 mg.or 5-carbon hydroxycarboxylic acids to yield five-(g-) or six-(d-) membered lactone rings. The daily per capita intakes of all the other flavouring agents in the group are in the range of 0. respectively. or oxidation or reduction of the side-chain and subsequent excretion as the glucuronic acid conjugate. The stability of the lactone ring in an aqueous environment is pH-dependent. hydroxylation of ring alkyl substituents producing polar metabolites that may be excreted. On the basis of the results of studies of structurally related lactones. The metabolic options open to lactones fused to alicyclic rings (Nos 1161–1166) include excretion as the open-chain hydroxycarboxylic acid derivative.4 and 1. The estimated daily per capita intakes in Europe and the USA for dihydrocoumarin are 1. Ten flavouring agents (Nos 1157–1166) in this group are expected to be metabolized to innocuous products. Step 2. In applying the Procedure for the Safety Evaluation of Flavouring Agents. In blood. The remaining 12 agents (Nos 1161– 1172) were assigned to structural class III. The daily per capita intake of each agent in Europe and in the USA is reported in Table 2. respectively. or oxidative degradation of the carboxylic acid side-chain to yield polar alicyclic or aromatic carboxylic acids that are excreted unchanged or in conjugated form. the four aliphatic lactones (Nos 1157–1160) in the group can be expected to hydrolyse to the corresponding hydroxycarboxylic acid. and then undergo b-oxidative cleavage to yield metabolites that are completely metabolized in the citric acid cycle. The evaluation of 56 . the Committee assigned four of the 16 agents (Nos 1157–1160) to structural class I.

these flavouring agents therefore proceeded via the A-side of the decision-tree. Limited metabolic data exist for the aromatic-fused ring lactones (Nos 1167–1172). The evaluation of these six flavouring agents therefore proceeded via the B-side of the decision tree. Step A3. The estimated daily per capita intakes of all four of the flavouring agents in structural class I (Nos 1157–1160) and six agents (Nos 1161–1166) in structural class III are below the threshold for concern (i.e. 1800 mg/day for class I and 90 mg/day for class III). The Committee concluded that these 10 substances would not be expected to be of safety concern at current estimated levels of intake as flavouring agents. Step B3. The estimated daily per capita intakes of four of the flavouring agents in structural class III are below the threshold for concern for their class (i.e. 90 mg/day). Accordingly, the evaluation of these four agents proceeded to step B4. The estimated daily per capita intakes of the remaining two substances in structural class III, dihydrocoumarin (No. 1171) and 6methylcoumarin (No. 1172), exceed the threshold of concern for their class (i.e. 90 mg/day). The estimated intake of dihydrocoumarin is 1415 mg/person per day in Europe and 1111 mg/person per day in the USA. The estimated intake of 6-methylcoumarin (No. 1172) is 298 mg/ person per day in Europe and 96 mg/person per day in the USA. In accordance with the Procedure, more extensive data are needed to perform a safety evaluation of flavouring agents exceeding the threshold for their structural class at step B3. Step B4. The NOELs of 5.42 and 6.55 mg/kg of body weight per day for males and females respectively for 3-propylidenephthalide (No. 1168) are 1000 times greater than its estimated intake of 0.3 mg/kg of body weight per day in Europe and 0.9 mg/kg of body weight per day in the USA. The NOELs for 3-propylidenephthalide are appropriate to evaluate 2-(4-methyl-2-hydroxyphenyl)propionic acid g-lactone (No. 1167), 3n-butylphthalide (No. 1169), and 3-butylidenephthalide (No. 1170) because these substances are structurally related and undergo similar pathways of metabolism. The NOELs of 5.42 and 6.55 mg/kg of body weight per day for males and females respectively for 3propylidenephthalide are 100 000 times greater than the estimated intake from use as a flavouring agent of 2-(4-methyl-2hydroxyphenyl)propionic acid g-lactone in the USA (0.03 mg/kg of body weight per day), 100 000 times greater than the estimated intake
57

of 3-n-butylphthalide in Europe (0.01 mg/kg of body weight per day) and in the USA (0.006 mg/kg of body weight per day), and 10 000 times greater than the estimated intake of 3-butylidenephthalide in Europe (0.2 mg/kg of body weight per day) and in the USA (0.1 mg/kg of body weight per day). The Committee concluded that these substances would not pose a safety concern at currently estimated levels of intake. Table 2 summarizes the evaluations of the members of this group. Consideration of flavouring agents with high intakes evaluated by the B-side of the decision-tree Dihydrocoumarin (No. 1171) More extensive data on metabolism and toxicity were considered to complete the safety evaluation of dihydrocoumarin (No. 1171). Dihydrocoumarin is a d-lactone fused to a benzene ring and is not a member of the class of aromatic coumarin derivatives. Dihydrocoumarin lacks a,b-unsaturation in the lactone ring, which is a key structural feature in the metabolism of coumarin. Coumarin is principally metabolized in humans and primates by electrophilic substitution (i.e. 7-hydroxylation), and in rats and several other species by epoxidation of the alkene function to form mainly 3hydroxycoumarin. The absence of a double bond in dihydrocoumarin precludes the formation of the reactive epoxide and subsequent metabolites. The metabolic fate of dihydrocoumarin closely resembles that of simple aliphatic d-lactones. Dihydrocoumarin and aliphatic lactones hydrolyse to the corresponding ring-opened hydroxyacids. Hydrolysis of dihydrocoumarin yields a substituted 3phenylpropanoic acid derivative that is expected to undergo either conjugation or side-chain oxidation to yield the corresponding benzoic acid derivative. In a 13-week study, groups of B6C3F1 mice received dihydrocoumarin in corn oil by gavage at doses of up to 1600 mg/kg of body weight, once daily, 5 days a week. No gross or microscopic lesions were observed in either sex at any dose, although body-weight gain was reduced and males and females at the highest dose and females at 800 mg/kg of body weight showed were changes in organ weights. The results of this study show that the NOEL for dihydrocoumarin is 400 mg dihydrocoumarin/kg of body weight per day for B6C3F1 mice. In a study of the potential carcinogenicity of dihydrocoumarin, B6C3F1 mice received dihydrocoumarin in corn oil by gavage at doses
58

up to 800 mg/kg of body weight, once daily, 5 days per week for 2 years. No significant differences in survival rates, final mean body weights or clinical findings were reported in the treated animals as compared with the controls. The only increase in neoplasia associated with the administration of dihydrocoumarin were increases in the incidences of hepatocellular adenoma and hepatocellular adenoma and carcinoma (combined), seen at all doses in females only. This effect reflects the high incidence of spontaneous liver tumours in this hybrid mouse and thus the heightened sensitivity to enhancement of liver neoplasia. In view of the nature of the findings, the Committee concluded that observations of hepatic neoplasms in this bioassay in mice are not relevant to the safety of dihydrocoumarin in humans at low levels of intake from use as a flavouring agent. In a 13-week study, groups of Fischer 344/N rats received dihydrocoumarin in corn oil by gavage at doses of up to 1200 mg/kg of body weight, once daily, 5 days per week. In males only, body weight was decreased at the highest dose. Changes in enzymes and other constituents of blood plasma were reported at doses ≥300 mg/kg of body weight. Increases in liver and kidney weights were observed at the two higher doses. Centrilobular hepatocellular hypertrophy, ranging in severity from minimal to mild, was reported in the livers of animals of each sex at doses of 300 mg/kg of body weight and above. No adverse treatment-related effects were observed in either male or female rats receiving 75 and 150 mg dihydrocoumarin/kg of body weight. In a study of the potential carcinogenicity of dihydrocoumarin, Fischer 344/N rats received doses of up to 600 mg/kg of body weight in corn oil by gavage, on 5 days per week for 2 years. A significant doserelated decrease in the survival of male rats, attributed to progressive degenerative nephropathy leading to renal failure, was reported after week 92. Nephropathy was reported in control and treated rats of each sex. Although the incidence of nephropathy was greater in male rats, the findings were significant only in females at the two higher doses. Microscopic examination revealed a statistically significant, dose-related increase in renal tubule hyperplasia in male rats only. A significant increase in the incidence of renal tubule adenomas was observed in males treated with 600 mg dihydrocoumarin/kg of body weight when compared to the control group, but there was no evidence of malignant renal tubule neoplasms in male rats at any dose. Increases in the incidence of renal tubule hyperplasia or renal tubule adenomas were not observed in female rats. The Committee concluded that the renal hyperplastic and neoplastic effects observed are sex- and species-specific and not dose-related, and that these effects
59

no effects were reported in three short-term studies in rats fed dihydrocoumarin in the diet. relative liver weights were significantly increased in the treated group compared with the controls. A dose-related increase in sister chromatid exchange was found in the same cell line. The NOEL was 300 mg/kg of body weight per day. The results of the assays for reverse mutation and unscheduled DNA synthesis were negative. no effects were reported in a long-term study in dogs fed dihydrocoumarin in the diet. A test for micronucleus formation in rats in vivo gave negative results.76% dihydrocoumarin (equivalent to 580 mg/kg of body weight). Dihydrocoumarin did not markedly affect the activities of carnitine acetyltransferase and palmitoyl-coenzyme A oxidation. In a study in rats fed diets containing 0. 1172). they did provide additional data that support the safe use of dihydrocoumarin as a flavouring agent at its current level of intake. As also noted at the thirty-fifth meeting. 6-Methylcoumarin (No. indicating that dihydrocoumarin is unlikely to be a rodent liver peroxisome proliferator. similar assays for forward mutation without metabolic activation produced negative results. Negative results were obtained in six out of seven studies of chromosomal aberration in Chinese hamster ovary cells. 1172) More extensive data on metabolism and toxicity were considered in order to complete the safety evaluation of 6-methylcoumarin (No. but no microscopic abnormalities were observed. Cytotoxicity was reported in a mouse lymphoma assay in the presence of an endogenous metabolic activation system. Dihydrocoumarin was tested in various assays for genotoxicity in vitro. It is anticipated that humans will metabolize 6-methylcoumarin via methyl group oxidation to the corresponding benzoic acid deriva60 . but this isolated positive result was not considered evidence for genotoxicity. Although it was not possible to determine a NOEL from these studies.reflect the sensitivity of the male rat kidney to chronic progressive nephropathy and neoplasia. As noted by the Committee at its thirty-fifth meeting. The Committee concluded that the data indicated that dihydrocoumarin is not genotoxic. The data obtained in this study were limited by the small number of animals tested. however.

necrosis and hepatitis. No significant differences in general health and behaviour. bradycardia. 61 .tive. or body weight and food consumption were reported in the treated animals as compared with the controls. serum biochemical or urinary parameters at any dose. including hypoactivity. were reported in animals of each sex at 600 mg/kg of body weight. No other changes were reported in haematological. 6-methylcoumarin was given to rats by gavage at doses of up to 1200 mg/kg body weight per day. No effects on organ weights. No treatment-related effects were reported in animals receiving a dose of 150 mg/kg of body weight per day. lachrymation. All rats receiving the highest dose of 1200 mg/kg of body weight and one male rat receiving 600 mg/kg of body weight died during week 1 of the study. and loss of the grasping reflex were reported at the highest dose only. macroscopic or microscopic examinations at any dose. Prostration. degeneration. which can also be readily excreted. In a 13-week study. or macroscopic or microscopic changes in the tissues were reported at any dose. Haematological examinations performed at the end of the study did not reveal any treatment-related effects. followed by excretion as the glucuronic acid conjugate. 1000 or 10 000 ppm (equivalent to 0. ataxia. Decreases in body weight were reported in males and females receiving 600 mg 6-methylcoumarin/kg of body weight. The clinical effects. When 6-methylcoumarin was administered daily by gavage to male and female B6C3F1 mice for 13 weeks at doses of up to 800 mg/kg of body weight. even in individuals exhibiting decreased activity of CYP2A6. these changes were not accompanied by any substance-related macroscopic observations. Necropsy of all animals receiving 1200 mg/kg of body weight revealed microscopic hepatic lesions that varied in the degree of congestion. groups of weanling Osborne-Mendel rats were fed diets containing 6-methylcoumarin at a concentration of 0. impaired righting reflex and decreased limb tone. bradypnoea. 6-Methylcoumarin may also undergo ring hydroxylation to form the corresponding 7-hydroxy metabolite. relative to controls. Increased mean absolute and relative liver weights were reported in males and females receiving doses of 300 and 600 mg/kg of body weight. no toxicologically significant changes were reported in clinical. hypoactivity. metabolism via the 3. however. 100 and 1000 mg 6-methylcoumarin/kg of body weight). In a 14-week study. A decrease in serum cholinesterase activity was reported in females receiving 300 mg/kg of body weight.4-epoxide is at most a minor pathway. At high doses. hypothermia.

In a long-term study. but in females only at the higher dose. 6-Methylcoumarin treatment increased mixed function oxidase activity (i. 6-Methylcoumarin was not genotoxic in a number of assays for reverse mutation with Salmonella typhimurium strains. In a 13-week study in rats given dihydrocoumarin. Hepatic effects in males and females receiving a dose of 750 mg/kg of body weight included fatty metamorphosis. This NOEL is about 5000 times more than the estimated per capita intake of dihydrocoumarin in Europe (24 mg/kg of body weight per day) and in the USA (19 mg/ kg of body weight per day). Osborne-Mendel rats were fed diets containing 6-methylcoumarin at concentrations up to 15 000 ppm (equivalent to 750 mg/kg of body weight). No increase in the frequency of mutation was observed in a test for sex-linked recessive lethal mutation in Drosophila melanogaster. no increases in plasma aminotransferase activity and no bile-duct hyperplasia or cholangiofibrosis were reported. very slight bile duct proliferation. and focal telangiectasis. but it did not markedly affect the activities of carnitine acetyltransferase and palmitoyl-coenzyme A oxidation. The results of a 13-week study conducted in rats fed diets containing 0. 7-ethoxycoumarin O-deethylase activity). The Committee concluded that the data indicated that 6-methylcoumarin is not genotoxic. no effects were reported in dogs fed diets containing 6-methylcoumarin at a concentration resulting in 50 mg/kg of body weight per day . marginally positive results were reported in a single assay. however. In rats.82% 6-methylcoumarin (corresponding to 695 mg/kg of body weight) revealed a slight vacuolation of hepatocytes in three out of eight treated animals. a NOEL of 150 mg/ kg of body weight per day was identified. Negative results were reported in a mouse lymphoma assay. the NOEL in the 2-year study by 62 . the positive results were not confirmed in a similar study.In a 2-year feeding study. No treatment-related effects were observed at doses up to and including 175 mg/kg of body weight per day in males and 375 mg/kg of body weight per day in females. Depression in growth rates was noted in males receiving doses of 375 or 750 mg/kg of body weight. A test for micronucleus formation in mice in vivo gave negative results in females and equivocal results in males. indicating that 6-methylcoumarin is unlikely to be a rodent liver peroxisome proliferator. No effects were reported in a limited study in dogs that received 6methylcoumarin at a dose of 200 mg/kg body weight per day in gelatine capsules for 2 or 4 weeks. The data obtained in these studies are limited because only one or two dogs were tested per group.e.

More extensive data on metabolism and toxicity were 63 . 1160 (g-dodecalactone and 2(3H)-furanone. Consideration of secondary components Three members of this group of flavouring agents (Nos 1158. dihydro-5-(2octenyl)-(Z)) were evaluated at the forty-ninth meeting. compounds that are structurally related to the secondary components of No.8-decanedione and 4-hydroxy-5.9-dimethyl 3. Consideration of combined intakes from use as flavouring agents All 16 agents in this group are expected to be efficiently metabolized and would not saturate metabolic pathways. Conclusions The Committee concluded that none of the 16 flavouring agents in this group of alicyclic. and were considered not to present a safety concern at current levels of intake. 1164 (3.4hexandione and b-ionone) were evaluated at the fifty-first meeting. a NOEL of 150 mg/kg of body weight per day was found. These NOELs are 10 000 times more than the estimated intake of dihydrocoumarin in Europe (24 mg/kg of body weight per day) and in the USA (19 mg/kg of body weight per day). None of the secondary components was considered to present a safety concern at current levels of intake. alicyclic-fused and aromatic-fused ring lactones would present safety concerns at the current estimated levels of intake. This NOEL is 30 000 times more than the estimated intake of 6-methylcoumarin in Europe (5 mg/kg of body weight per day) and in the USA (2 mg/kg of body weight per day). The secondary components of No. the secondary components of No. Understanding of their metabolism and the available data on toxicity led the Committee to conclude that the safety of dihydrocoumarin and 6-methylcoumarin would not be expected to present a safety concern at current levels of intake (Table 2). 1164 (2.gavage was 300 mg/kg of body weight per day for dihydrocoumarin. The secondary components of No. Evaluation of all the data indicated no safety concern associated with combined intake. 1160. and 1164) have minimum assay values of <95%. On this basis. 1164 were considered not to present a safety concern at current levels of intake. However. In a 13-week study in rats. 1158 (heptan-1-ol) and No. Information on the safety of the secondary components of these three compounds is summarized in Annex 4 (Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less).6-oxo b-ionone) have not been evaluated previously.

acids and esters The Committee evaluated a group of 26 a. 1178. a. 1195–1197). This group included 12 dienals (Nos 1173. a trienal (No. five dienols (Nos 1174. broccoli. 1185–1187. 1175. sorbic acid) in its capacity as an antimicrobial preservative at the seventeenth meeting (Annex 1.b-unsaturated. 1198). The Committee had evaluated one member of the group.1. 4. reference 32). all of which contain unsaturation in the 2. a. 1176. tea and beer. and are only consumed to a minor extent as added flavouring agents. (E. a dienoic acid (No. 1171) in accordance with the application of the Procedure in the case of flavouring agents with high intakes evaluated by the B-side of the decision-tree. 1179. 1191–1194). when an ADI of 0–25 mg/kg of body weight was established. di. A monograph summarizing the safety data on this group of flavouring agents was prepared. alicyclic. 1184 and 1189). 1182. Twenty-one of the 26 flavouring agents in this group of flavouring agents have been reported to occur naturally in food. The per capita intakes of all the other flavouring 64 .5-position.b-unsaturated flavouring agents (see Table 3) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. 1190).E)2. 1180.2 Aliphatic. roast chicken.and trienals and related alcohols. grapes.4-trans-decadienal is approximately 20 mg in Europe and 70 mg in the USA. 1176) and seven related esters (Nos 1177. The estimated combined per capita daily intake of the 12 dienals is approximately 40 mg in Europe and 120 mg in the USA. 1181. Approximately 50–60% of the total annual volume of production of the aliphatic linear dienals in Europe and the USA is accounted for by 2-trans. 1190. They have been detected in apples.considered in the evaluations of dihydrocoumarin (No.b-Unsaturated aldehydes are formed endogenously by lipid peroxidation of polyunsaturated fatty acids (PUFA) or can be consumed as naturally-occurring constituents of food. Estimated daily per capita intake The total annual volume of production of the 26 flavouring agents in this group is approximately 1000 kg in Europe and 1500 kg in the USA. and the per capita daily intake of 2-trans. 1183. 1188. 1).4-trans-decadienal (No.4-hexadienoic acid (No. Other data on the toxicity and metabolism of these ring lactones were consistent with the results of the safety evaluation. linear.3-position and some of which contain saturation in the 4. 1172) and 6methylcoumarin (No.

b CAS No. acids and esters used as flavouring agentsa. linear. alicyclic.1 USA: 0.6-Nonadien-1-ol 7786-44-9 HO 1184 NR See note 2 No safety concern NR See note 1 No safety concern Nona-2-trans-6-cisdienal 557-48-2 H O 1186 65 No Europe: 2 USA: 1 No Europe: 7 USA: 24 . and structure Comments Flavouring agent No.and trienals and related alcohols. Step A3 Does intake exceed the threshold for human intake?c Step A4 Is the flavouring agent or are its metabolites endogenous? NR See note 3 Conclusion based on current intake Structural class I (E. a.Table 3 Summary of results of the safety evaluation of aliphatic.007 See note1 No safety concern 2.1 USA: ND See note 4 No safety concern Ethyl sorbate 2396-84-1 NR O O 1178 No Europe: 59 USA: 3 NR See note 4 No safety concern 1182 56767-18-1 H O 2-trans.E)-2. di.4-Hexadienoic acid 110-44-1 O HO 1176 No Europe: ND USA: 6 NR See footnote d Methyl sorbate 689-89-4 O O 1177 No Europe: 0.b -unsaturated.6-transOctadienal No Europe: 0.

Step A3 Does intake exceed the threshold for human intake?c Step A4 Is the flavouring agent or are its metabolites endogenous? NR See note 1 No Europe: ND USA: 0.4 See note 4 No safety concern Propyl 2.Z)-2.9 USA: ND NR See note 4 No safety concern .7decatrienoate 78417-28-4 O O 1193 No Europe: ND USA: 0. and structure Comments Flavouring agent No.6-Nonadien1-ol acetate No Europe: ND USA: 18 No Europe: ND USA: 1 NR See note 4 No safety concern Methyl (E)-2-(Z)-4decadienoate 4493-42-9 O O 1191 See note 4 No safety concern Ethyl trans-2-cis-4decadienoate 3025-30-7 O O 1192 No Europe: 34 USA: 3 NR NR See note 4 No safety concern Ethyl 2.007 NR Conclusion based on current intake 1187 17587-33-6 H O 2-trans-6-transNonadienal No safety concern 1188 68555-65-7 O O (E.4.4decadienoate 84788-08-9 O O 1194 No Europe: 0.66 Table 3 (continued) CAS No.

4-hexadien-1-ol when used as a flavouring agent Yes.4hexadienal is >1 million times the estimated daily intake of (E. The NOEL of 15 mg/kg bw per day for the related substance trans.4-heptadienal when used as a flavouring agent See note1 No safety concern 67 .1 USA: 0.trans-2.trans-2.4 See note 2 No safety concern trans.4Hexadienal 142-83-6 H O 1175 No Europe: 1 USA: 0.4-Hexadien1-ol 111-28-4 HO 1174 No Europe: ND USA: 0.02 NR See note1 No safety concern O H 1197 2. The NOEL of 15 mg/kg bw per day for the related substance trans.4hexadienal is >10 000 times the estimated daily intake of 2.2-trans-6-cisDodecadienal 21662-13-5 No Europe: 0.trans-2.6 USA: 0.trans-2.4-Heptadienal 4313-03-5 O H 1179 No Europe: 4 USA: 23 Yes.4-Pentadienal 764-40-9 See note 1 H O 1173 No Europe: 0. The NOEL of 15 mg/kg bw per day for the related substance trans.E)-2.trans-2.4-hexadienal when used as a flavouring agent Yes.2 No safety concern (E.E)-2.4-pentadienal when used as a flavouring agent Yes. The NOEL of 15 mg/kg bw per day is >100 000 times the estimated daily intake of trans.1 See note 1 No safety concern 2.4hexadienal is >1 million times the estimated daily intake of 2.

E)-2.trans-2.4hexadienal is >1 million times the estimated daily intake of trans.4Octadienal 30361-28-5 H O 1181 No Europe: 0.4-Nonadien-1-ol 62488-56-6 HO 1183 No Europe: ND USA: 26 See note 2 No safety concern 2.trans-2. The NOEL of 33.trans-2. The NOEL of 33.E)-2.4-Octadien1-ol 18409-20-6 HO 1180 No Europe: ND USA: 18 trans.7 Yes.4-octadienal when used as a flavouring agent Yes.4-transdecadienal is >10 000 times the estimated daily intake of 2.68 Table 3 (continued) CAS No. The NOEL of 15 mg/kg bw per day for the related substance trans. Step A3 Does intake exceed the threshold for human intake?c Step A4 Is the flavouring agent or are its metabolites endogenous? See note 2 Conclusion based on current intake No safety concern (E.7 USA: 0.9 mg/kg bw per day for the related substance 2-trans.007 See note1 No safety concern 2.4-Nonadienal 6750-03-4 H O 1185 No Europe: 2 USA: 0. The NOEL of 15 mg/kg bw per day for the related substance trans.4-octadien-1-ol when used as a flavouring agent Yes.4hexadienal is >10 000 times the estimated daily intake of (E.4-transdecadienal is >1 million times See note 1 No safety concern .trans-2.4-nonadien-1-ol when used as a flavouring agent Yes. and structure Comments Flavouring agent No.9 mg/kg bw per day for the related substance 2-trans.

4-transdecadienal is >1 million times the estimated daily intake of trans.4-transDecadienal No Europe: 22 USA: 70 See note 1 No safety concern 2.4-transdecadienal is >10 000 times the estimated daily intake of (E. The NOEL of 33.E)-2.E)-2.9 mg/kg bw per day for the related substance 2-trans.4-transdecadienal when used as a flavouring agent Yes.4-decadien-1-ol when used as a flavouring agent Yes.9 mg/kg bw per day for the related substance 2-trans. The NOEL of 33.4trans-decadienal is >10 000 times the estimated daily intake of 2-trans. The NOEL of 33.4 See note 1 No safety concern trans.4-transdecadienal is >100 000 times the estimated daily intake of 2.4-nonadienal when used as a flavouring agent Yes.4Dodecadienal 21662-16-8 H O 1196 No Europe: 0.4-Undecadienal 13162-46-4 O H 1195 No Europe: 4 USA: 0.4-Decadien1-ol 18409-21-7 See note 2 HO 1189 No Europe: ND USA: 26 No safety concern 1190 25152-84-5 H O 2-trans.7 USA: 0.9 mg/kg bw per day for 2-trans.1 the estimated daily intake of 2. The NOEL of 33.9 mg/kg bw per day for the related substance 2-trans.4dodecadienal when used as a flavouring agent See note 1 No safety concern 69 .trans-2.4-undecadienal when used as a flavouring agent Yes.(E.trans-2.

may undergo glutathione conjugation and excretion as mercapturic acid derivatives. 1190. Notes to Table 3: 1. Oxidized to aldehydes and acids. 1195. Alternately.009 Yes. The combined per capita intakes of flavouring agents in structural class I are 138 mg per day in Europe and 221 mg per day in the USA. The NOEL of 33 mg/kg bw per day is >1 million times the estimated daily intake of 2-trans-4-cis-7-cistridecatrienal when used as a flavouring agent Conclusion based on current intake No safety concern 2-trans-4-cis-7-cisTridecatrienal 13552-96-0 O H 1198 CAS: Chemical Abstract Service. 3. d An ADI of 0-25 mg/kg bw was established for (E. and structure Comments Flavouring agent No. b Step 2: Thirteen flavouring agents (Nos 1176–1178. 1179–1181. NR: not required for evaluation because consumption of the substances was determined to be of no safety concern at step A3 of the decision-tree. a Step 1: All of the flavouring agents in this group are in structural class I. 1182.E)-2. 1185. reference 32). and 1197) in this group are expected to be metabolized to innocuous products. and 1198) in this group cannot be predicted to be metabolized o innocuous products. The evaluation of these flavouring agents therefore proceeded via the A-side of the decision-tree. Oxidized to acids. 1183. The a. The evaluation of these 13 flavouring agents therefore proceeded via the B-side of the decision-tree. 1189. 1184. 1186–1188. . 1191–1194. 1196. 2. and this was maintained at the present meeting. which metabolize completely in the fatty acid b-oxidation pathway.70 Table 3 (continued) CAS No. Step A3 Does intake exceed the threshold for human intake?c Step A4 Is the flavouring agent or are its metabolites endogenous? See note 1 No Europe: 0. c The threshold for human intake for structural class I is 1800 mg per day.3 USA: 0. 4.b-unsaturated dienals and related alcohole (Nos 1173–1175. All intake values are expressed in mg per day.4-hexadienoic acid by the Committee at its seventeenth meeting (Annex 1. ND: no intake data reported. Use of the chemical as a flavouring agent is subsumed in the ADI. Hydrolysed to corresponding alcohols and acids. which may undergo b-oxidative cleavage and complete metabolism via the tricarboxylic acid cycle. Undergo b-oxidative cleavage and complete metabolism via the tricarboxylic acid cycle. followed by complete metabolism in the fatty acid pathway or the tricarboxylic acid cycle.

The unsaturated alcohols are oxidized successively to the corresponding aldehydes and carboxylic acids. 1189. However. 1196. 1179–1181. distribution. Under conditions of glutathione depletion and oxidative stress.007–24 mg/day. aliphatic esters are hydrolysed rapidly to their component alcohols and carboxylic acids by classes of enzymes known as carboxylesterases in the intestinal mucosa. In applying the Procedure for the Safety Evaluation of Flavouring Agents to the 26 flavouring agents in this group. 1184. and at high cellular concentrations. including the fatty acid pathway and tricarboxylic acid cycle.agents in the group are in the range of 0. Absorption.bunsaturated 2.b-unsaturated aldehydes are safely metabolized in the high-capacity b-oxidation pathway or via glutathione conjugation. 71 . 1186– 1188. metabolic evidence indicates that low concentrations of a. 1183. which may undergo b-oxidative cleavage and complete metabolism via the tricarboxylic acid cycle. metabolism and elimination In general. 1195. It is anticipated that humans will metabolize dienals and trienals by oxidation to the corresponding acids. and 1197) in this group are expected to be metabolized to innocuous products. 1191–1194. Once hydrolysed. The evaluation of these flavouring agents therefore proceeded via the A-side of the decision-tree. An alternate minor pathway may involve conjugation of the unsaturated aldehyde to glutathione. most values being at the lower end of this range. which participate in fundamental biochemical pathways. 1182. 1185. Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. The a. the resulting aliphatic alcohols and carboxylic acids are absorbed into the portal circulation. the Committee assigned all of them to structural class I. The daily per capita intake of each agent in Europe and in the USA is reported in Table 3.4-dienals and alcohol precursors (Nos 1173–1175. Thirteen flavouring agents (Nos 1176–1178. Step 2. and to induce apoptosis. and 1198) cannot be predicted to be metabolized to innocuous products and the evaluation of these 13 flavouring agents therefore proceeded via the B-side of the decision-tree. followed by excretion as the mercapturic acid derivative. 1190.b-unsaturated aldehydes have been shown to form adducts with DNA nucleotides. a. to cause cytohistopathology.

1180) because these alcohols will be oxidized to the corresponding aldehydes and subsequently undergo metabolism in a similar manner to the dienals. because of their similar metabolic pathways. Step B4. 1181). 1189).4-nonadienal (No. 1196).4octadien-1-ol (No.4-pentadienal (No. 1191– 1194. the NOEL of 33 mg/ kg of body weight per day identified from a 4-week study in rats provides an adequate margin of safety (>1 000 000) in relation to the known levels of intake of this agent.4-nonadien-1-ol (No. 1185). 1179). This NOEL is also appropriate for the structurally related agents 2. The NOEL of 33. The Committee concluded that these substances would not be expected to be of safety concern at their currently estimated levels of intake as flavouring agents. 1190).E)-2. 2.4-transdecadienal (No. The estimated daily per capita intakes in Europe and the USA of the remaining 13 flavouring agents in this group that cannot be predicted to be metabolized to innocuous products are also below the threshold of concern for structural class I (1800 mg/ day).E)-2. 1184. provides an adequate margin of safety (>10 000) in relation to the known levels of intake of this agent. 1195) and trans. 2. 1182.e. 1173).4trans-decadienal is also appropriate for the structurally related substances 2.4-heptadienal (No.4-decadien-1-ol (No. Accordingly.E)-2.4-undecadienal (No.4-hexadienal is also appropriate for the structurally related (E.4-hexadienal (No.trans-2.4-dodecadienal (No. Step B3. 1175) administered by gavage in a 14week study in rats provides an adequate margin of safety (>100 000) in relation to the known levels of intake of this agent.4-octadienal (No. The NOEL of 15 mg/kg of body weight per day for trans. The Committee noted that 2. the evaluation of these 13 agents proceeded to step B4. 1186–1188. The NOEL for 2-trans.trans-2.4-hexadien-1-ol (No. 1800 mg/day). 1174). The estimated daily per capita intakes in Europe and the USA of the 13 flavouring agents in this group that are metabolized to innocuous products (Nos 1176–1178. 2. 1198). 1175) induced forestomach hyperplasia and squamous cell tumours in rats and mice 72 .4-trans-hexadienal (No. and (E. because these agents are all dienals which will undergo oxidation and subsequent metabolism via similar metabolic pathways. The NOEL for trans.9 mg/kg of body weight per day for 2-trans. For 2-trans-4-cis-7-cis-tridecatrienal (No. and trans. and 1197) are below the threshold for concern for class I (i.trans-2. (E. 1183).trans-2. identified from a 14-week study in rats treated by gavage.Step A3.

of each sex. Mice and rats in the bioassays developed forestomach hyperplasia following corn oil gavage. but was inactive in tests carried out in vivo. could theoretically deplete concentrations of GSH . this substance may be genotoxic under some conditions.trans-2. Accordingly. An IARC Working Group concluded that when evaluating the relevance for human cancer of the induction of forestomach tumours in rodents. Agents that only produce tumours of the forestomach in rodents after prolonged treatment. but this is not believed to be the basis for its effects in the rodent forestomach. at sufficiently high concentrations. concentrations of intracellular replenishable GSH (approximately 1–10 mM) are 73 . Thus. the Committee concluded that the appearance of forestomach tumours in the 2-year bioassays in rodents in which trans. Trans.trans-2. Table 3 summarizes the evaluations of the 26 a. Consideration of combined intakes from use as flavouring agents Although the flavouring agents evaluated in this group are not converted to a common metabolite. The forestomach was the only site of increased neoplasia in treated animals.4-hexadienal was administered at high concentration by gavage is of no relevance to humans. Therefore.b-unsaturated aldehydes. they are subject to conjugation with reduced glutathione (GSH). may be of less concern to humans since human exposure to such agents would need to surpass time-integrated dose thresholds in order to elicit the carcinogenic response. There was evidence of injury to the forestomach epithelium attributable to exposure and this is believed to be the primary cause of the development of neoplasia. simultaneous consumption of the a. under normal conditions. However.b-unsaturated flavouring agents in this group. resulting in lipid peroxidation. the experimental conditions of exposure should be considered. reflecting the sensitivity of the forestomach to irritation. through non-DNA-reactive mechanisms. This is a common finding in USA National Toxicology Program bioassays in which a high concentration of an irritating material suspended in corn oil is delivered by gavage into the forestomach every day for 2 years. and a low incidence of adenomas was observed in mice.4-hexadienal gave positive results in some tests for genotoxicity in vitro. The conditions of exposure during oral administration are unusual in that physical effects may cause high local concentrations of test substances in the forestomach and prolonged exposure of the epithelium.

4-nonen-1-ol was considered not to present a safety concern at current intake levels. 1180. since the a.E)-2. 1192.b-unsaturated aldehydes being ingested as flavouring agents.4-hexadienoic acid (No. 1190 and 1196). 1196 and 1198) have minimum assay values of <95%. The Committee concluded that none of the flavouring agents in this group would pose a safety concern at the currently estimated levels of intake. 1176. On this basis. 1176). Consideration of secondary components Ten members of this group of flavouring agents (Nos 1179. at the levels of a. Additionally.1 mg/kg of body weight per day) from its use as a flavouring agent is below the individual ADI (0–25 mg/kg of body weight) established previously by the Committee (Annex 1. or have metabolites that are substrates for the fatty acid cycle. The estimated current intake of (E. 1180.4-nonadien-1-ol) was evaluated at the present meeting. None of the secondary components was considered to present a safety concern at current levels of intake. Information on the safety of the secondary components of these 10 compounds is summarized in Annex 4 (Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less). and in consideration of the constant replenishment of GSH by biosynthesis.sufficient to detoxify the quantities of a. One of the secondary components of No. and 1198).b-unsaturated aldehydes used as flavouring agents. it is unlikely that all foods containing these flavouring agents would be consumed concurrently on a daily basis. 1185 (2.bunsaturated aldehydes provide similar flavouring characteristics. 1189. the Committee considered that the combined intake of these flavouring agents does not present a safety concern. 1183. 1191. while two of the secondary components of No. sorbic acid) (0. it is expected to be oxidized and completely metabolized in the fatty acid cycle. 2.4-hexadienoic acid (No. reference 33). the secondary components were expected to share the same metabolic fate as the primary flavouring agents (Nos 1179. 1189–1192.4-nonen-1ol) has not been evaluated previously. Conclusions The Committee retained the previously established ADI of 0–25 mg/ kg of body weight for (E. 1185 (2. 1185. 1190 (acetone and isopropanol) were evaluated at the fifty-first meeting. however. It was noted that other data on the toxicity and metabolism of 74 . The other secondary component of No. which are subsequently excreted as carbon dioxide and water (Nos 1183. In all cases. 1185.E)-2. Therefore.

reference 131). linalool and linalyl acetate. acids. aldehydes. 1230) have been reported to occur naturally in foods. expressed as citral. geranyl acetate. 1). citronellol. was established for citral. 1225) were both evaluated by the Committee at its eleventh meeting (Annex 1.the flavouring agents in the group were consistent with the results of the safety evaluation. A monograph summarizing the safety data on this group of flavouring agents was prepared. A group ADI of 0–0. the group ADI was maintained. kumquats. that a long-term study was required for at least one member of this group. when conditional ADIs of 0–0. They have been detected in fruits such as raspberries. reference 50) as part of a group of terpenoid flavouring agents. 1223–1225. at its fifty-first meeting (Annex 1. aldehydes. the Committee evaluated a group of 26 geranyl. Likewise. and linalyl acetate on the basis of their clearly-defined metabolism. 4. The Committee maintained. citronellol (No. citronellyl. were allocated. aldehydes. 75 . 1212. The Committee concluded that there were no safety concerns for any of the 26 substances under the low levels of intake arising from their use as flavouring agents and maintained the group ADI for citral. citronellol.3 Aliphatic branched-chain saturated and unsaturated alcohols. At its forty-ninth meeting (Annex 1. bananas.25 mg/kg of body weight and 0–1 mg/kg of body weight respectively. linalool. geranyl acetate. and linalyl acetate. Two-year studies of carcinogenicity had been conducted for a mixture of two of these esters. 1219) and citral (No. Citronellol and citral were re-evaluated at the twenty-third meeting of the Committee (Annex 1. Twenty-four of the 32 flavouring agents (Nos 1199–1202.5 mg/kg of body weight.1. 1228. and rhodinyl esters formed from branched-chain terpenoid alcohols and aliphatic acyclic linear and branched-chain carboxylic acids by the Procedure. 1208–1210. however. 1213. rapid excretion. reference 14). reference 137) when the Committee re-evaluated linalool and linalyl acetate. The substances that occur naturally in the highest abundance are the monoterpene primary alcohols. 1204–1206. and low toxicity in shortterm studies. 1217–1221. 1215. linalool. and carboxylic acids. as well as in many alcoholic beverages. acids. 1227. neryl. and myrtle berries. and related esters The Committee evaluated a group of flavouring agents comprising 32 aliphatic branched-chain saturated and unsaturated alcohols. and related esters (see Table 4) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. strawberries. geranyl acetate and citronellyl acetate. including geranyl acetate. The Committee had previously evaluated two members of this group.

including ginger. There is no evidence for accumulation in the body. together with the other alcohols. coriander. faeces. 76 . brandy and rum. 1224). mustard. Shorter branched-chain aliphatic alcohols. The daily intakes per capita of all the other flavouring agents in the group are estimated to be in the range of 0. all of the flavouring agents in this group will eventually be either completely oxidized or oxidized to polar metabolites which are excreted primarily in the urine. most values being below 50 mg. and citral (No. w-1 and b-oxidation. distribution. citral accounts for approximately 73% of the total annual volume of production in Europe and 80% in the USA. sage and thyme. and acids undergo b-oxidative cleavage to yield intermediates of the amino acid and/or fatty acid metabolic pathways. and elimination The four esters in this group can be expected to be hydrolysed by esterases to the corresponding alcohols and carboxylic acids. the latter substances. aldehydes. Once formed. after absorption the substances can be expected to be distributed rapidly throughout the body. metabolized. nerol (No. 1220). respectively. As chain length and substitution increase.including beer. the alcohols and aldehydes undergo a combination of w-. metabolism. geraniol (No. and excreted as polar metabolites in the urine.01–945 mg. in citrus fruits and in many spices and spice oils. and expired air. Estimated daily per capita intake The total annual volume of production of the 32 flavouring agents in this group is approximately 66 000 kg in Europe and 66 000 kg in the USA. Therefore. are readily absorbed from the gastrointestinal tract. 1219). On the basis of the results of a study with citral. whisky. however. citronellal (No. More than 95% of the total annual volume of production in Europe and the USA is accounted for by citronellol (No. and selective dehydrogenation and hydration to yield polar acidic metabolites. Of these. cinnamon. chamomile. These intermediates are completely metabolized to CO2 via the tricarboxylic acid cycle. acids and aldehydes in this group. The daily per capita intake of each agent in Europe and in the USA is reported in Table 4. 1223). Absorption. wine. The estimated daily per capita intakes of citral in Europe and the USA are 6849 mg and 6990 mg. They are most abundant. 1225). The substances in this group share common metabolic pathways.

aldehydes. acids. and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the flavouring agent or are its metabolites endogenous? Step A5 Adequate margin of safety for the flavouring agent or related substance? NR See note 1 NR No Europe: ND USA: 35 No Europe: 5.Table 4 Summary of results of the safety evaluations of aliphatic branched-chain saturated and unsaturated alcohols.4 USA: 3.2 NR 1201 1115-11-3 See note 1 H No safety concern O 3-Methyl-2-butenal H 1202 No Europe: 3.5 NR 107-86-8 NR See note 1 No safety concern O 1203 O 7563-33-9 No Europe: 18 USA: 16 NR See note 2 No safety concern Ammonium isovalerate (ammonium salt of isovaleric acid) HO 77 . and related esters used as flavouring agentsa Flavouring agent Comments on predicted metabolism No.9 USA: 0. CAS No.8 NR NR NR NR Conclusion based on current intake Structural class I (+/-) 2-Methyl-1butanol 1199 137-32-6 No safety concern HO 3-Methyl-2-buten1-ol 1200 556-82-1 See note 1 No safety concern HO 2-Methyl-2-butenal No Europe: 0.7 USA: 0.

01 NR NR Conclusion based on current intake No safety concern 3-Methylcrotonic acid O 1204 541-47-9 HO trans-2-Methyl-2butenoic acid No Europe: 4.9 USA: 1.2 NR NR See note 1 No safety concern H .5 USA: 45 589-66-2 See note 3 No safety concern 2-Methylallyl butyrate NR O O 1207 No Europe: ND USA: 0.3 USA: 0.6 O 1205 80-59-1 See note 2 HO No safety concern Isobutyl 2butenoate NR NR O O 1206 No Europe: 0.78 Table 4 (continued) Flavouring agent Comments on predicted metabolism See note 2 No. CAS No. and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the flavouring agent or are its metabolites endogenous? Step A5 Adequate margin of safety for the flavouring agent or related substance? NR NR No Europe: 121 USA: 0.2 7149-29-3 NR See note 3 No safety concern 4-Methyl-2pentenal O 1208 5362-56-1 No Europe: 0.

1 USA: 0.01 1214 25409-08-9 NR NR See note 4 No safety concern O H 79 .4-Dimethyl-2pentenoic acid No Europe: 0.2 1209 623-36-9 O H 2-Methyl-2pentenoic acid No Europe: 42 USA: 20 NR NR See note 2 O 1210 3142-72-1 No safety concern HO 2.1 7779-81-9 NR See note 3 No safety concern 2-Butyl-2-butenal No Europe: ND USA: 0.1 USA: 0.2-Methyl-2pentenal NR NR See note 1 No safety concern No Europe: 4 USA: 0.1 NR NR 1211 66634-97-7 See note 2 No safety concern HO O 2-Methylheptanoic acid NR NR No Europe: 17 USA: 6 O 1212 1188-02-9 See note 2 HO No safety concern Isobutyl angelate NR O O 1213 No Europe: 0.

80 Table 4 (continued) Flavouring agent Comments on predicted metabolism See note 4 No.9 1217 73757-27-4 NR See note 1 No safety concern H O 4-Ethyloctanoic acid OH 1218 No Europe: ND USA: 4 16493-80-4 NR NR See note 2 No safety concern O .01 Conclusion based on current intake No safety concern 2-Isopropyl-5methyl-2hexenal O 1215 35158-25-9 H 2-Ethyl-2-heptenal No Europe: 0.01 USA: 0.3 USA: 0. and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the flavouring agent or are its metabolites endogenous? Step A5 Adequate margin of safety for the flavouring agent or related substance? NR NR No Europe: 0.1 NR NR 1216 10031-88-6 See note 4 No safety concern H O 2-Methyl-2-octenal NR No Europe: ND USA: 7. CAS No.

4 NR NR 1222 6812-78-8 See note 4 HO No safety concern Geraniol No Europe: 640 USA: 315 No Europe: 290 USA: 171 NR No 1223 106-24-1 See note 4 HO No safety concern Nerol 1224 106-25-2 NR See note 4 No safety concern HO 1225 H 5392-40-5 Yes Europe: 6849 USA: 6990 See note 4 * Citral (Mixture of the trans and cis isomers geranial and neral) O Yes. The NOEL of 60 mg/kg of bodyweight per day for citral is >500 times the estimated daily 81 .7-Dimethyl-6octenoic acid No Europe: 3.dl-Citronellol No Europe: 370 USA: 0.1 USA: 0.2 NR NR 502-47-6 No safety concern HO Rhodinol No Europe: 53 USA: 8.5 NR NR See note 4 * NR NR See note 4 1219 106-22-9 HO Citronellal H 1220 No Europe: 945 USA: 324 NR NR See note 4 106-23-0 No safety concern O 1221 O 3.

5 .82 Table 4 (continued) Flavouring agent Comments on predicted metabolism No. CAS No. 11-dodecatrienal O H No safety concern 1228 O 19317-11-4 NR See note 4 3.6.11-Trimethyl2.2 No Europe: ND USA: 0.7.1 USA: 0.6. and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the flavouring agent or are its metabolites endogenous? Step A5 Adequate margin of safety for the flavouring agent or related substance? Conclusion based on current intake O H 8-Ocimenyl acetate NR O O 1226 No Europe: ND USA: 7.7 NR NR 197098-61-6 intakes of 114 mg/kg of bodyweight in Europe and 117 mg/kg of bodyweight in the USA when used a flavouring agent NR See note 4 No safety concern 1227 No Europe: 5.5 NR 60066-88-8 See note 4 2.6-Dimethyl-10methylene-2.10dodecatrienal 12-Methyltridecanal H H No safety concern NR NR See note 1 No safety concern 1229 75853-49-5 O No Europe: ND USA: 0.

reference 50). The combined per capita intake of flavouring agents in structural class I is 9382 mg per day in Europe and 8732 mg per day in the USA. expressed as citral. The acid may undergo partial b-oxidation. 4. 83 . a All of the flavouring agents in this group are expected to be metabolized to innocuous products. geranyl acetate.5 mg/kg of body weight. then participates in the pathway cited in notes 1 and 2. * A group ADI of 0–0. and linalyl acetate by the Committee at its 23rd meeting (Annex 1. 2. which was maintained at the present meeting.6 1230 4602-84-0 See note 4 HO No safety concern CAS: Chemical Abstracts Service. Metabolized primarily via the b-oxidation pathway yielding shorter chain carboxylic acids that are subsequently metabolized to CO2 via the tricarboxylic acid pathway. linalool. If unsaturation is present. was established for citral. NR: not required for evaluation because consumption of the agent was determined to be of no safety concern at Step A3 of the Procedure. ND: no intake data reported. Primarily oxidized to corresponding carboxylic acid that may enter the b-oxidation pathway yielding shorter chain carboxylic acids that are subsequently metabolized to CO2 via the tricarboxylic acid pathway. the polar polyoxygenated metabolites may also form hydrogenation or hydration metabolites. Oxidized to corresponding carboxylic acid. All intake values are expressed in mg/day. Use of citronellol and citral as flavouring agents is subsumed in the group ADI. Hydrolysed to the corresponding alcohol and carboxylic acid. citronellol.Farnesol NR NR No Europe: 9 USA: 2. be excreted or undergo w oxidation to yield polar polyoxygenated metabolites that are excreted free or conjugated primarily in the urine. 3. b The threshold for human intake for structural class I is 1800 mg/day. Notes to Table 4: 1.

Step 2. 1225) from a 2-year study of carcinogenicity is approximately 500 times greater than the estimated intake of citral from its use as a flavouring agent in Europe (114 mg/kg of body weight per day) and in the USA (117 mg/kg of body weight per day). The estimated daily per capita intakes of 31 of the 32 flavouring agents are below the threshold of concern for structural class I (1800 mg). However. 1211. Step A3. citral (No. the agents in this group are expected to be metabolized efficiently and the available metabolic pathways would not be saturated. The evaluation of all agents in this group therefore proceeded via the A-side of the decision-tree. Consideration of secondary components Seven members of this group of flavouring agents (Nos 1209. 1219–1223) have assay values of <95%. saturated and unsaturated alcohols. Table 4 summarizes the evaluations of the 32 aliphatic branchedchain. Consideration of combined intakes from use as flavouring agents In the unlikely event that all 32 of these flavouring agents were to be consumed concurrently on a daily basis.Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. The Committee concluded that the safety of these 31 flavouring agents raises no concern at their currently estimated levels of intake as flavouring agents. One of the agents. the estimated combined per capita intake would exceed the human intake threshold for structural class I (1800 mg per day). The daily per capita intake of citral is 6849 mg in Europe and 6990 mg in the USA. The NOEL of 60 mg/kg of body weight per day for citral (No. In applying the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. Step A4. aldehydes. the Committee assigned all of the flavouring agents in this group to structural class I. The Committee therefore concluded that citral would not pose a safety concern at the currently estimated level of intake. Evaluation of all the data indicated no safety concern associated with combined intake. the evaluation of citral proceeded to step A4. and related esters (Nos 1199–1230) in this group. Step A5. acids. exceeds the threshold of concern for class I. The evaluation of citral therefore proceeded to step A5. Accordingly. All the flavouring agents in this group are expected to be metabolized to innocuous products. Information on the safety of the secondary components of these seven compounds is summarized in Annex 4 (Summary of the safety evaluation of secondary compo84 . Citral is not endogenous in humans. 1). 1225).

On this basis. 1220 was considered to present a safety concern on the basis of current intake levels. The Committee concluded that the safety of the flavouring agents in this group of aliphatic branched-chain 85 . geranyl acetate. The Committee noted that the estimated combined intake for citronellol (Table 4). 1211 (4-methyl-2-methylenevaleric acid) has not been evaluated previously. 1221 (citronellal) was evaluated at the present meeting. none of the secondary components of Nos. for citral. while three other secondary components (linalool. and citronellyl acetate) were evaluated at the fifty-first. nerol. and linalyl acetate. and acetic acid. linalool. One of the secondary components of No. The secondary component of No. None of the secondary components of No. reference 137). geranyl acetate (Annex 1. 1221–1223 was considered to present a safety concern on the basis of current levels of intake. and geranyl acetate esters) are expected to be hydrolysed to the corresponding terpene alcohols (citronellol.nents for flavouring agents with minimum assay values of 95% or less). and geraniol). which was evaluated at the forty-ninth meeting. 1220 (eucalyptol) was evaluated at the present meeting. reference 137) is approximately 0. Some of the secondary components of Nos. expressed as citral. The secondary components of No. citronellol. Two of the secondary components of No. citral (Table 4).15 mg/kg of body weight per day in the USA. reference 131). none of the secondary components was considered to present a safety concern at current intake levels. however. while the remaining secondary component (citronellyl acetate) was evaluated at the fifty-ninth meeting. 1219 (geraniol and citronellal) were evaluated at the present meeting. One of the secondary components of No.15 mg/kg of body weight in Europe and the USA) is less than the group ADI. and linalyl acetate (Annex 1. isopulegol. fifty-fifth and fifty-ninth meetings. 1209 (propionaldehyde and propionic acid) were evaluated at the forty-ninth meeting and were considered not to present a safety concern at current levels of intake.20 mg/kg of body weight per day in Europe and 0. and therefore does not exceed the group ADI. a structurally related substance (isovaleric acid) was evaluated at the forty-ninth meeting and considered not to present a safety concern at current levels of intake. which were evaluated at the present meeting. Conclusions The Committee maintained the previously established group ADI of 0–0. and considered not to present a safety concern at current levels of intake.5 mg/kg of body weight. It was also noted that even the total combined daily intake of all 32 flavouring agents under evaluation (approximately 0. 1221–1223 (citronellyl. neryl. linalool (Annex 1.

1. 1). 1251) 86 . 1234). distribution. 1248–1255) have been reported to occur naturally in foods. These agents have not been evaluated previously by the Committee. More than 90% of the total annual volume of production in Europe and >75% in the USA is accounted for by eucalyptol (No. The open-chain aliphatic compounds can be expected to undergo O-dealkylation to yield the corresponding aldehyde and alcohol. metabolism and elimination The aliphatic ethers in this group are either open-chain (Nos 1231– 1232) or cyclic compounds (Nos 1233–1240). Benzyl butyl ether (No. Twenty-three of the 29 flavouring agents (Nos 1231–1239.003– 241 mg/day. The estimated daily per capita intake of eucalyptol in Europe and the USA is 1439 mg and 1954 mg. 1234) and anisole (No. 1241) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. acids. followed by complete oxidation in the fatty acid pathway and tricarboxylic acid cycle. Estimated daily per capita intake The total annual volume of production of the 29 flavouring agents in this group is approximately 11 000 kg in Europe and 19 000 kg in the USA.4 Aliphatic and aromatic ethers The Committee evaluated a group of 29 aliphatic and aromatic flavouring agents (see Table 5) that included eucalyptol (No. They have been detected in fruits. alcoholic beverages. Absorption. A monograph summarizing the safety data on this group of flavouring agents was prepared. reference 53). The Committee noted that all of the available data on toxicity and metabolism of the flavouring agents in the group were consistent with the results of the safety evaluation. The daily per capita intake of each agent in Europe and in the USA is reported in Table 5. vegetables. respectively.saturated and unsaturated alcohols. 1256) were evaluated for specifications only at the twenty-fourth meeting (Annex 1. Most of the aromatic flavouring agents in this group have single benzene ring structures with an ether group and one or more simple saturated (Nos 1241–1250 and 1252–1254) or unsaturated (No. oil and tea. The daily per capita intakes of the other flavouring agents in the group range from 0. 1253) and dibenzyl ether (No. The alicyclic ethers can be expected to undergo either ring hydroxylation or sidechain oxidation followed by conjugation with glucuronic acid and excretion in the urine. 1241–1246. 4. aldehydes. cheese. and related esters would not raise concern at the currently estimated levels of intake.

and structure Step A3 b Does intake exceed the threshold for human intake? Flavouring agent Comments on predicted metabolism No.2 NR See note 3 No safety concern 1-Methyl-3methoxy-4isopropylbenzene O 1246 1076-56-8 No Europe: 2 USA: 0.5 USA: 15 NR See note 2 No safety concern 2.03 USA: 0.1 NR NR See note 3 No safety concern 87 .Table 5 Summary of results of the safety evaluations of aliphatic and aromatic ethers used as flavouring agentsa CAS No. Step A4 Is the substance or its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? NR See note 1 NR Conclusion based on current intake Structural class I Anisole O 1241 100-66-3 No Europe: 0.4-Dimethylanisole O 1245 6738-23-4 No Europe: ND USA: 0.06 NR NR See note 1 No safety concern p-Methylanisole O 1243 104-93-8 No Europe: 0.01 NR NR No safety concern o-Methylanisole O 1242 578-58-5 No Europe: 3 USA: 0.

1 USA: 0. Step A4 Is the substance or its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? NR NR Conclusion based on current intake No safety concern Carvacryl ethyl ether No Europe: 0.2Dimethoxybenzene No Europe: ND USA: 20 NR NR See note 3 No safety concern 1249 151-10-0 No Europe: 5 USA: 2 O See note 3 m-Dimethoxybenzene O No safety concern p-Dimethoxybenzene O O 1250 150-78-7 No Europe: 18 USA: 7 NR NR See note 3 No safety concern Structural class II sec-Butyl ethyl ether O 1231 2679-87-0 No Europe: 8 USA: 0.88 Table 5 (continued) CAS No.02 O 1247 4732-13-2 1248 91-16-7 NR NR O O 1. and structure Step A3 b Does intake exceed the threshold for human intake? Flavouring agent Comments on predicted metabolism See note 3 No.3 NR NR See note 4 No safety concern .

9 USA: 2 NR NR See note 4 No safety concern No safety concern No safety concern NR NR See note 5 1.7 No Europe: 0.01 USA: 8 NR Yes.2.4-Cineole O 1233 470-67-7 No Europe: 5 USA: 146 NO Yes Europe: 1439 USA: 1954 Eucalyptol O 1234 470-82-6 Nerol oxide NR O 1235 1786-08-9 No Europe: 1 USA: 0. The NOEL of See note 5 >32 mg/kg of body weight per day for eucalyptol is approximately 1000 times the estimated daily intakes of 24 mg/kg of body weight in Europe and 33 mg/kg of body weight in the USA when used as a flavouring agent NR See note 5 No safety concern 2.1-Ethoxy-3-methyl-2 butene O 1232 22094-00-4 No Europe: 0.6-Trimethyl-61236 7392-19-0 vinyltetrahydropyran O NR See note 5 No safety concern 1237 16409-43-1 O Tetrahydro-4-methyl2-(2-methylpropen1-yl) pyran No Europe: 4 USA: 0.2 NR NR See note 5 No safety concern 89 .

01 NR NR See notes 3 and 6 No safety concern Methyl phenethyl ether O 1254 3558-60-9 NR See notes 3 and 6 No safety concern .90 Table 5 (continued) CAS No.003 USA: 2 NR NR See notes 3 and 6 No safety concern Benzyl butyl ether O 1253 588-67-0 No Europe: ND USA: 0. and structure Step A3 b Does intake exceed the threshold for human intake? Flavouring agent Comments on predicted metabolism See note 5 No.1 Cycloionone No Europe: ND USA: 2 NR NR 1239 5552-30-7 See note 5 No safety concern O Benzyl ethyl ether NR O 1252 539-30-0 No Europe: 0. Step A4 Is the substance or its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? NR NR Conclusion based on current intake No safety concern Theaspirane O 1238 36431-72-8 No Europe: 2 USA: 0.02 No Europe: 31 USA: 0.

1 No safety concern p-Propylanisole NO O 1244 104-45-0 Yes Europe: 23 USA: 114 See note 7 No safety concern 3.9)) tridecane No Europe: 1 USA: 0.9-Tetramethyl13-oxatricyclo (8.5. The NOEL of 300 mg/kg of body weight for the related substance p-propenylanisole (trans anethole) is >100 000 times the daily intakes of p-propylanisole in Europe (0.0(4.5.4-Dimethoxy-1vinylbenzene O O 1251 6380-23-0 No Europe: ND USA: 0.1240 3738-00-9 NR NR See note 5 O Structural class III 1.3.4 mg/kg of body weight per day) and in the USA (2 mg/kg body weight) when used as a flavouring agent NR See notes 3 and 6 No safety concern Diphenyl ether O 1255 101-84-8 NR NR See note 8 No safety concern 91 .0.01 No Europe: 14 USA: 5 NR Yes.

Step A4 Is the substance or its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? No Conclusion based on current intake Dibenzyl ether O 1256 103-50-4 Yes Europe: 0. when used as a flavouring agent NR See note 9 No safety concern .01 mg/kg of bodyweight in Europe and 4 mg/kg of bodyweight in the USA. respectively. The NOEL of 196 mg/kg of bodyweight per day (females) and >620 mg/kg bw per day (males) for dibenzyl ether is >10 000 000 and >10 000 times the estimated daily intakes of 0.01 NR Yes. and structure Step A3 b Does intake exceed the threshold for human intake? Flavouring agent Comments on predicted metabolism No.92 Table 5 (continued) CAS No.6 USA: 241 See notes 3 No safety and 6 concern b-Naphthyl methyl ether O 1257 93-04-9 No Europe: ND USA: 0.

Metabolized primarily by m-hydroxylation with O-demethylation 3. The combined per capita intakes of flavouring agents in structural class III are 40 mg per day in Europe and 386 mg per day in the USA. 5. followed by conjugation with glucuronic acid and excretion in the urine 6. Metabolized by O-demethylation. Metabolized by cytochrome P450-catalysed O-dealkylation to the corresponding alcohol and aldehyde. Step 2: All of the flavouring agents in this group are expected to be metabolized to innocuous products. Notes to Table 5: 1.b-Naphthyl ethyl ether No Europe: ND USA: 4 NR NR See note 9 O 1258 93-18-5 No safety concern b-Naphthyl isobutyl ether No Europe: 1 USA: 2 NR NR O 1259 2173-57-1 See note 9 No safety concern CAS: Chemical Abstracts Service. respectively. The combined per capita intakes of flavouring agents in structural class II are 1491 mg per days in Europe and 2115 mg per day in the USA. a and w-1 oxidation of the side chain and side chain degradation 8. and o-hydroxylation is the minor pathway 2. followed by complete oxidation in the fatty acid pathway and tricarboxylic acid cycle. b The threshold for human intake for structural classes I. Metabolized by ring hydroxylation 7. Oxidized by cytochrome P450 isoenzymes to polar metabolites. The combined per capita intakes of flavouring agents in structural class I are 29 mg per day in Europe and 44 mg per day in the USA. Metabolized by O-demethylation 4. Metabolized primarily by p-hydroxylation with O-demethylation. All intake values are expressed in mg per day. 540 mg/day and 90 mg/day. Metabolized by ring hydroxylation followed by conjugation with glucuronic acid and excretion 9. ND: no intake data reported. NR: not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3 of the Procedure. Excreted as a glucuronic acid conjugate with the methyl ether linkage intact a 93 . II and III are 1800 mg/day.

1) to the 29 flavouring agents in this group. None of these three flavouring agents is endogenous in humans. or side-chain oxidation). 1256). The evaluation of all agents in this group therefore proceeded via the A-side of the decision-tree. In applying the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. sulfate or glycine. 1244) and dibenzyl ether (No. depending on the location of the substituents. The NOEL of >32 mg/kg of body weight per day for eucalyptol (No. Step A5. Others in this group have two aromatic rings.e. These aromatic ethers can be expected to be metabolized by one or more of three pathways (ring hydroxylation. Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. The daily intake of eucalyptol per capita has been reported to be 1439 mg in Europe and 1954 mg in the USA.6 mg in Europe and 241 mg in the USA. The daily intake per capita of p-propylanisole is 23 mg in Europe and 114 mg in the USA. O-dealkylation. All the flavouring agents in this group are expected to be metabolized to innocuous products. Step A4. Twelve flavouring agents (Nos 1231–1239. Accordingly. the evaluation of these agents proceeded to step A4. The Committee concluded that these 26 substances would not be expected to be of safety concern when used as flavouring agents at currently estimated levels of intake. Step 2. 1234) is approximately 1000 times greater than the estimated intake of eucalyptol from its use as a flavouring agent in Europe 94 . and then conjugated with glucuronic acid. 1255–1259) were assigned to structural class III. 1244. The evaluation of these substances therefore proceeded to step A5. and two agents in structural class III. 1234). The estimated daily per capita intakes of all nine of the flavouring agents in structural class I. Some have dual methoxy groups (Nos 1248–1251). p-propylanisole (No. which are either separate (Nos 1255 and 1256) or fused (Nos 1257–1259). 1800 mg for class I. 11 of the 12 agents in structural class II. Step A3. eucalyptol (No.side-chains. and 1252–1254) were assigned to structural class II and the remaining eight (Nos 1240. the Committee assigned nine (Nos 1241–1243 and 1245–1250) to structural class I. and six of the eight agents in structural class III are below the threshold of concern (i. The daily intake per capita of dibenzyl ether is 0. Intake of one of the agents in structural class II. 540 mg for class II and 90 mg for class III). 1251. exceed the thresholds of concern for class II and III.

01 mg/kg of body weight per day in Europe and 4 mg/kg of body weight per day in the USA). 1256) provides a margin of safety that is 50 000 times greater than the highest estimated intake of dibenzyl ether from its use as a flavouring agent (0. a margin of safety of approximately 120 existed between this intake and the NOEL of >32 mg/kg of body weight per day established in a chronic study conducted in rats. the secondary component in benzyl butyl ether.4-cineole (No. thus it was not possible to estimate chronic intake. the secondary component in 1. The Committee noted. Information on the safety of the secondary components of these three compounds is summarized in Annex 4 (Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less). 1. reference 138). was evaluated at a previous meeting (Annex 1. 25). The NOEL of 300 mg/kg of body weight per day for ppropenylanisole. provides a margin of safety that is approximately 150 000 times greater than the highest estimated intake of p-propylanisole (No. The NOEL of 196 mg/kg of body weight per day for dibenzyl ether (No. The secondary components in these two flavouring agents were considered not to present a safety concern. 1244) from its use as a flavouring agent (0. 1. 1253). Evaluation of all the data indicated no safety concern associated with combined intake. 1 The Committee was informed that certain products. 1233) and benzyl butyl ether (No.4-cineole. Table 5 summarizes the evaluations of the 29 aliphatic and aromatic ethers (Nos 1231–1259) in this group. such as lozenges/hard candies. reference 122). Consideration of combined intakes from use as flavouring agents All 29 agents in this group are expected to be metabolized efficiently and the available metabolic pathways would not be saturated. however. may contain high levels of eucalyptol and noted that theoretical estimates of daily per capita intake of eucalyptol could be as great as 16 mg.4 mg/kg body weight per day in Europe and 2 mg/kg of body weight per day in the USA). have a minimum assay value of <95%. was evaluated at the present meeting. that even at an intake per capita of 16 mg per day.8-Cineole (No. while benzyl alcohol (No. Data on the frequency of use of such products were not available to the Committee.(24 mg/kg of body weight per day) and in the USA (33 mg/kg of body weight per day)1. 1234). identified by the Committee (Annex 1. Consideration of secondary components Two members of this group. The Committee therefore concluded that the safety of these agents raises no concern at their currently estimated levels of use. 95 .

distribution. and pork. commonly recognized as isoeugenol derivatives. A monograph summarizing the safety data on this group of flavouring agents was prepared. ethoxy. by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig.1. raw fatty fish. 1266).5 Hydroxypropenylbenzenes The Committee evaluated a group of flavouring agents that included nine hydroxy. Other data on the toxicity and metabolism of these aromatic and aliphatic ethers were consistent with the results of the safety evaluation. The estimated daily per capita intake of isoeugenol is approximately 120 mg in Europe and 40 mg in the USA. Three of the nine flavouring agents (Nos 1260. and > 65% of the total annual volume of production in the USA is accounted for by propenylguaethol (No. They have been detected in blueberries. The estimated daily per capita intake of isoeugenyl methyl ether is approximately 130 mg in Europe and 130 mg in the USA. Absorption.or alkoxy-substituted propenylbenzenes (see Table 6). Estimated daily per capita intake The total annual volume of production of the nine flavouring agents in this group is approximately 2000 kg in Europe and 4100 kg in the USA. mushrooms. The daily per capita intakes of the other flavouring agents in the group range from 0. The estimated daily per capita intake of propenylguaethol is approximately 40 mg in Europe and 350 mg per day in the USA. 96 . More than 80% of the total annual volume of production in Europe is accounted for by isoeugenol (No. 1). The daily per capita intake of each agent in Europe and in the USA is reported in Table 6. 1265 and 1266) have been reported to occur naturally in foods. 1260) and isoeugenyl methyl ether (No. metabolism and elimination Six (Nos 1260–1265) of the nine flavouring agents contain a free phenolic OH group or are simple phenolic esters.009–11 mg. ginger. These agents have not been evaluated previously by the Committee. or benzoxy substituents on the para (p) position and a methoxy substituent on the meta (m) position. The remaining three agents (Nos 1266–1268) are propenyl benzene derivatives that contain a methoxy. with most being <1 mg. 1264). 4.Conclusions The Committee concluded that none of the flavouring agents in this group of aliphatic and aromatic ethers would raise a safety concern at the currently estimated levels of intake.

Table 6 Summary of results of the safety evaluations of hydroxypropenylbenzenesa Flavouring agent Comments on predicted metabolism No.7 USA: 11 93-29-8 NR See note 3 No safety concern Isoeugenyl phenylacetate O O O 1263 No Europe: ND USA: 0. and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the substance or are its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? NR See note 1 NR Conclusion based on current intake Structural class I Isoeugenol No Europe: 117 USA: 43 1260 97-54-1 HO O No safety concern Isoeugenyl formate NR NR O O H 1261 No Europe: ND USA: 0.3 120-24-1 NR NR See note 4 No safety concern 97 . CAS No.2 7774-96-1 See note 2 No safety concern O Isoeugenyl acetate NR O O O 1262 No Europe: 0.

and structure Step A3 b Does intake exceed the threshold for human intake? Step A4 Is the substance or are its metabolites endogenous? Step A5 Adequate margin of safety for substance or related substance? NR NR No Europe: 44 USA: 354 Conclusion based on current intake Propenylguaethol HO O 1264 94-86-0 No safety concern 4-Propenyl-2.6dimethoxyphenol NR NR O 1265 No Europe: ND USA: 2 20675-95-0 See note 1 No safety concern Structural class III HO O Isoeugenyl methyl ether No O 1266 Yes Europe: 128 USA: 129 93-16-3 See note 5 No safety concern O Yes.Table 6 (continued) 98 Flavouring agent Comments on predicted metabolism See note 1 No. CAS No. The NOEL of 6 mg/kg of body weight per day for isoeugenol methyl ether is >1000 times the estimated daily intakes of 2 mg/kg of bodyweight per day in Europe and the USA when used as a flavouring agent .

b The threshold for human intake for structural classes I and III are 1800 mg/day and 90 mg/day. respectively. Hydrolysed to isoeugenol and formic acid. Hydrolysed to isoeugenol and acetic acid. The combined per capita intake of flavouring agents in structural class III is 129 mg per day in Europe and 130 mg per day in the USA. NR: not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3 of the procedure. 4. a Step 2: All of the flavouring agents in this group are expected to be metabolized to innocuous products. Notes to Table 6: 1. ND: no intake data reported.1267 NR NR See note 5 O O Isoeugenyl ethyl ether No Europe: ND USA: 0. which is oxidized to CO2 and H2O. 5. Hydrolysed to isoeugenol and phenylacetic acid. which is endogenous in humans and excreted as the glutamine conjugate. 3. 99 . Detoxicated primarily by O-demethylation at the (m) or (p)-methoxy substituent to yield the corresponding phenol followed by excretion in the urine as the sulfate or glucuronic acid conjugate. The combined per capita intake of flavouring agents in structural class I is 162 mg per day in Europe and 411 mg per day in the USA.009 7784-67-0 No safety concern Isoeugenyl benzyl ether NR NR O O 1268 No Europe: 1 USA: 1 120-11-6 See note 5 No safety concern CAS: Chemical Abstracts Service. Detoxication primarily by conjugation of the phenolic OH group with sulfate or glucuronic acid and excretion mainly in the urine 2. All intake values are expressed in mg per day. which is absorbed from the gastrointestinal tract and acts as a precursor for synthesis of biomolecules.

Upon hydrolysis the product. Step A3. The evaluation of all agents in this group therefore proceeded via the A-side of the decision-tree. exceeds the threshold of concern. Step A4. The estimated daily per capita intakes of all six of the flavouring agents in structural class I and two of the three agents in structural class III are below the threshold of concern (i. Esters of isoeugenol (Nos 1261–1263) are hydrolysed in vivo by carboxylesterases. no adverse effects were observed in rats at a 100 . In applying the Procedure for the Safety Evaluation of Flavouring Agents to the above-mentioned flavouring agents. Accordingly. Isoeugenyl methyl ether is not endogenous in humans. Step 2. the Committee assigned six of the nine flavouring agents (Nos 1260–1265) to structural class I. 1266). primarily in the urine.Isoeugenol derivatives containing a phenolic OH group (Nos 1260. 1800 mg for class I and 90 mg for class III). is conjugated and excreted while the component carboxylic acids are metabolized in well-recognized biochemical pathways. One of the agents in structural class III. 1266) was identified from a 28-day study in rats fed diets containing isoeugenyl methylether. 1264 and 1265) are rapidly absorbed from the gastrointestinal tract and are metabolized principally in the liver via conjugation of the phenolic hydroxy group with sulfate or glucuronic acid. The Committee concluded that the safety of these eight flavouring agents raises no concern at their currently estimated levels of intake. Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. The conjugates are subsequently excreted. All the flavouring agents in this group are expected to be metabolized to innocuous products. isoeugenol. A NOEL of 100 mg/kg of body weight per day for isoeugenyl methylether (No. Step A5. reference 137). the evaluation of isoeugenyl methyl ether proceeded to step A4. The remaining three flavouring agents (Nos 1266– 1268) were assigned to structural class III. isoeugenyl methyl ether (No. The alkoxypropenylbenzene derivatives (Nos 1266–1268) in this group primarily undergo O-demethylation of either the (m) or (p)methoxy substituent to yield the corresponding isoeugenol derivative that is then excreted as the sulfate or glucuronic acid conjugate (Annex 1.e. The evaluation therefore proceeded to step A5. The daily per capita intake of isoeugenyl methyl ether is 128 mg in Europe and 129 mg in the USA. In another study of longer duration (13 weeks).

pending review of a 90-day study of ethyl 2-methyl-3. It was concluded that 41 of the 42 substances in this group were of no safety concern at currently estimated levels of intake. Conclusions The Committee concluded that the flavouring agents in this group of hydroxypropenylbenzenes would not be of safety concern at the currently estimated levels of intake. acids and related esters : additional compounds The Committee evaluated 20 flavouring agents that included linear and branched-chain aliphatic. aldehydes. ethyl 2-methyl-3.6 Linear and branched-chain aliphatic. 4.dietary intake of 6 mg isoeugenyl methyl ether/kg of body weight per day. Evaluation of all the data indicated no safety concern associated with combined intake. Other data on the toxicity and metabolism of these hydroxypropenylbenzenes were consistent with the results of the safety evaluation. An addendum to the monograph summarizing the safety data on this group of flavouring agents was prepared. The NOEL of 6 mg/kg of body weight per day was >1000 times the estimated intake of isoeugenyl methyl ether from its use as a flavouring agent in Europe and in the USA (2 mg/kg of body weight per day in each case). unsaturated.1. The remaining six substances are linear unsaturated 101 . Table 6 summarizes the evaluations of nine hydroxypropenylbenzenes (Nos 1260–1268). the Committee concluded that isoeugenyl methyl ether is not expected to be of safety concern at currently estimated levels of use. unsaturated. reference 137). Fourteen of the 20 agents evaluated at the present meeting are esters formed from linear or branched-chain unsaturated alcohols or carboxylic acids.4-pentadienoate in the diet. acids and related esters (see Table 7) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. 353) was deferred. unconjugated alcohols. Consideration of combined intakes from use as flavouring agents All agents in this group are expected to be metabolized efficiently and the available metabolic pathways would not be saturated. 1). The Committee had previously evaluated 42 other members of this chemical group of flavouring agents at the fifty-first meeting (Annex 1. aldehydes. unconjugated alcohols. On the basis of these data.4pentadienoate (No. The evaluation of one substance.

and structure Comments Flavouring agent Step A3 c Does intake exceed the threshold for human intake? See note 1 Conclusion based on current intake Structural class I Isoprenyl acetate 1269 5205 07 2 O O No USA: 11 Europe: 9 See note 1 No safety concern 4-Pentenyl acetate 1270 1576-85-8 O O No USA: 4 Europe: 4 No safety concern 3-Hexenal 1271 4440-65-7 O H No USA: 53 Europe: 29 See note 2 No safety concern 3-Hexenyl formate 1272 2315 09 5 O O H No USA: 18 Europe: 14 See note 1 No safety concern Ethyl 5-hexenoate 1273 54653-25-7 O O No USA: 4 Europe: 4 See note 1 No safety concern . aldehydes.102 Table 7 Summary of results of safety evaluations of linear and branched-chain aliphatic.b No. acids and related estersa. unsaturated. unconjugated alcohols. CAS No.

1 Europe: 0.1 See note 1 No safety concern cis-3-Hexenyl tiglate 1277 67883-79-8 No USA: 70 Europe: ND See note 1 No safety concern O O cis-3-Hexenyl valerate 1278 35852-46-1 O O No USA: 9 Europe: 7 See note 1 No safety concern 3-Hexenyl 2-hexenoate 1279 53398-87-1 O O No USA: 0.07 See note 1 No safety concern 103 .2 Europe: 0.cis-3-Hexenyl propionate 1274 33467-74-2 See note 1 No safety concern O O No USA: 18 Europe: 14 cis-3-Hexenyl isobutyrate 1275 41519-23-7 O O No USA: 18 Europe: 14 See note 1 No safety concern (Z )-3-Hexenyl (E )-2-butenoate 1276 65405-80-3 O O No USA: 0.

104 Table 7 (continued) No.9 Europe: ND See note 3 No safety concern (E)-3-(Z )-6-Nonadien-1-ol 1284 56805-23-3 No USA: 0.9 Europe: ND OH See note 3 No safety concern . CAS No.Z )-3-6-Nonadien-1-ol 1283 53046-97-2 OH No USA: 0. and structure Comments Flavouring agent Step A3 c Does intake exceed the threshold for human intake? No USA: 2 Europe: ND See note 3 Conclusion based on current intake No safety concern (Z )-4-Hepten-1-ol 1280 6191-71-5 OH Ethyl-cis-4-heptenoate 1281 39924-27-1 O O No USA: 4 Europe: 4 No USA: 4 Europe: ND O See note 1 No safety concern (Z )-5-Octenyl propionate 1282 196109-18-9 O See note 1 No safety concern (Z.

The aldehyde is oxidized to the corresponding carboxylic acid. which is completely metabolized in the fatty acid and tricarboxylic acid pathways to carbon dioxide and water. The primary alcohol is oxidized to the corresponding aldehyde and carboxylic acid. The cumulative per capita intake for the amended group as a whole including the 42 agents in the original evaluation and the 20 additional substances is 5744 and 2760 mg per day in Europe and the USA. reference 137). which is completely metabolized in the fatty acid and tricarboxylic acid pathways to carbon dioxide and water. The ester is expected to undergo hydrolysis to the corresponding primary alcohol and carboxylic acid. Forty-two flavouring agents in this same congeneric group were previously evaluated by the Committee (Annex 1. The alcohol is oxidized to the corresponding aldehyde and carboxylic acid.4 Europe: 0. 2. which is subsequently oxidized in the fatty acid pathway and the tricarboxylic acid cycle. 3. respectively. c The threshold for human intake for structural class I is 1800 mg/day. ND: no intake data reported.(E)-3-(Z)-6-Nonadien-1-ol acetate 1285 211232-05-6 See note 1 O O No USA: 18 Europe: ND See note 2 No safety concern 9-Decenal 1286 39770-05-3 H O No USA: 0. All intake values are expressed in mg/day. The combined per capita intake of the flavouring agents is 103 mg per day in Europe and 239 mg per day in the USA. Notes to Table 7: 1.7 No USA: 1 Europe: 2 O OH No safety concern 4-Decenoic acid 1287 26303-90-2 See note 4 No safety concern cis-4-Decenyl acetate 1288 67452-27-1 O O No USA: 2 Europe: 2 See note 1 No safety concern CAS: Chemical Abstracts Service. a 105 . 4. The carboxylic acid is completely metabolized in the fatty acid and tricarboxylic acid pathways. b Step 2: All of the flavouring agents in this group are expected to be metabolized to innocuous products.

unsaturated. raw pork. No. thyme). and transported to the liver via the lymphatic system (12). spearmint oil. Long chain (C > 8) aldehydes (No. peppermint oil. cis-3-hexenyl tiglate. distribution. 1278) and by 3-hexenal (No. After the action of 3-hydroxyacyl coenzyme A epimerase has converted cis isomers to trans isomers. aldehydes. a variety of herbs and spices (e. acids and related esters is approximately 720 kg in Europe and 1400 kg in the USA (Table 8). and the double bond has been isomerized from the 3. linear unsaturated carboxylic acids enter the fatty acid pathway and are cleaved to yield acetyl or propionyl coenzyme A and subsequently completely metabolized to carbon dioxide and water in the tricarboxylic acid cycle. 1285 and 1288) in this group can be expected to hydrolyse to the corresponding unsaturated aliphatic alcohol and carboxylic acid (6–10). None of these agents had been evaluated previously. is accounted for by four cis-3-hexenyl esters (cis-hexenyl isobutyrate. beer. No. Once absorbed. 1275. scotch whisky. coriander. 1271-1278. They have been detected in fruit. chervil. 106 . the linear and branched-chain unsaturated primary alcohols are rapidly absorbed from the gastrointestinal tract (11) and oxidized to their corresponding aldehydes.g. hop oil. 1286) are readily absorbed as micelles. and 1287) in this group have been reported to occur as natural components of foods. olives. No.to the 2-position by enoyl coenzyme A isomerase (13). 1277. unconjugated alcohols. Approximately 63% and 70% of the total annual volume of production in Europe and in the USA. 1284. Fourteen of the 20 flavouring agents (Nos 1269.alcohols and aldehydes. 1281. aldehydes are oxidized to their corresponding unsaturated carboxylic acids. The daily per capita intake and poundage of each agent in Europe and in the USA are reported in Tables 7 and 8. Once formed. cis-hexenyl propionate. 1281. 1286. No. respectively. Estimated daily per capita intake The total annual volume of production of these 20 linear and branched-chain aliphatic. 1272-1279. 1270. and cis-hexenyl valerate. 1282. 1271). metabolism and elimination The aliphatic esters (Nos 1269. 1274. 1283. deposited in chylomicrons or low density lipoproteins. roast beef and chicken (5). chamomile oil. Absorption. black tea.

2 0.04 0.3 0.Table 8 Annual volumes of production of linear and branched-chain aliphatic.2 0.06 0.02 0.002 0.004 Isoprenyl acetate (1269) Europe USA 4-Pentenyl acetate (1270) Europe USA 3-Hexenal (1271) Europe USA 3-Hexenyl formate (1272) Europe USA Ethyl 5-hexenoate (1273) Europe USA cis-3-Hexenyl propionate (1274) Europe USA cis-3-Hexenyl isobutyrate (1275) Europe USA (Z)-3-Hexenyl (E)-2-butenoate (1276) Europe USA 6 + 0.001 0.5 0.13 + 0.05 Most recent annual volume (kg)a 60 60 9 11 0.2 0. unsaturated.07 0.1 107 NA .07 0.07 0.2 0. acids and related esters used as flavouring agents in Europe and the USAa Intakeb mg/day mg/kg bw per day Intake of alcohol equivalents mg/kg bw per dayc Annual volume in naturally occurring foods (kg)d 0.2 0.002 4 4 29 53 14 18 4 4 14 18 14 18 25 25 200 300 100 100 25 25 100 100 100 100 1 1 Consumption ratioe Substance (No.06 0.2 0.1 0.) NA - NA + NA + NA + 0.9 0.2 0.003 0.4 f NA 0. unconjugated alcohols.2 0.2 0.1 0.3 0. aldehydes.1 0.3 0.02 0.1 0.

9 50 50 0.07 ND 0.05 0.108 Table 8 (continued) Intakeb mg/day mg/kg bw per day Intake of alcohol equivalents mg/kg bw per dayc Annual volume in naturally occurring foods (kg)d Most recent annual volume (kg)a ND 400 ND 70 ND 1 0.06 0.05 0.05 - NA Icis-3-Hexenyl tiglate (1277) Europe USA cis-3-Hexenyl valerate (1278) Europe USA 3-Hexenyl 2-hexenoate (1279) Europe USA (Z)-4-Hepten-1-ol (1280) Europe USA Ethyl cis-4-heptenoate (1281) Europe USA (Z)-5-Octenyl propionate (1282) Europe USA (Z.7 NA .001 ND 0.5 0.1 0.1 0.) NA 8 0.02 7 9 0.001 + 0.07 ND 0.9 ND 0.5 0.2 - NA - NA + NA 0.01 0.02 0.03 0.001 0.6-Nonadien-1-ol (1283) Europe USA (E)-3-(Z)-6-Nonadien-1-ol (1284) Europe USA 48.Z)-3.6 + 9.1 ND 2 4 4 ND 4 ND 0.07 0.5 ND 9 25 25 ND 23 ND 5 ND 5 Consumption ratioe Substance (No.01 ND 0.0005 0.

03 0.3 0. where body weight = 60 kg. which are the maximum amounts of flavouring agent estimated to be used annually in both Europe and the USA by the manufacturer at the time the material was proposed for flavour use (15). “eaters only”) = 32 ¥ 106 for Europe and 26 ¥ 106 for the USA. kg) f Engel and Tressl (1983) a 109 . but no quantitative data.006 + 0. +.03 0. not available. The volumes cited are the anticipated annual volumes. kg)/(most recently reported volume as a flavouring agent. -.ND 100 ND 18 ND 0.2 0. where population (10%. kg) x (1 ¥ 109 mg/kg)/(population x survey correction factor ¥ 365 days].7 0. Slight variations may occur from rounding.01 0.6 for Europe and USA representing the assumption that only 60% of the annual flavour volume was reported (15). Intake (mg/kg bw per day) calculated as follows: [(mg/person per day)/body weight]. c Calculated as follows: (molecular weight alcohol/molecular weight ester) ¥ daily per capita intake (“eaters only”) ester d Quantitative data for the USA reported by Stofberg and Grundschober (16) e The consumption ratio is calculated as follows: (annual consumption in food.04 0.4 1 2 2 2 103 239 5 2 10 10 15 10 717 1351 NA NA 5772 577.03 0.02 0. not reported to occur naturally in foods. ND.2 (E)-3-(Z)-6-Nonadien-1-ol acetate (1285) Europe USA 9-Decenal (1286) Europe USA 4-Decenoic acid (1287) Europe USA cis-4-Decenyl acetate (1288) Europe USA Total Europe USA - NA NA. no intake data reported. The correction factor = 0. b Intake (mg/person per day) was calculated as follows: [(annual volume.02 0. reported to occur naturally in foods (5).

1271. all 62 agents in this group are expected to be metabolized efficiently and the available metabolic pathways would not be saturated. however. 1271 (trans-2-hexenal) has not been previously evaluated.e. 1279. The secondary component of No. aldehydes. In applying the Procedure for the Safety Evaluation of Flavouring Agents to the 20 flavouring agents in this group. Both cis-3-hexenol and 3hexenal (No. The Committee concluded that the safety of these 20 flavouring agents raises no concern when they are used at their currently estimated levels of intake. 1800 mg). The estimated daily per capita intakes of all 20 of the flavouring agents in structural class I are below the threshold of concern (i. Table 7 summarizes the evaluations of 20 linear and branched-chain aliphatic. based on a NOEL of 30 mg/kg of body weight per day from a 13-week study of oral administration in rats. Step 2. the Committee assigned all of them (Nos 1269–1288) to structural class I (14). unconjugated alcohols. However. the estimated combined daily per capita intake would exceed the human intake threshold for class I (1800 mg). The evaluation of all agents in this group therefore proceeded via the A-side of the decision-tree. Step A3. All the flavouring agents in this group are expected to be metabolized to innocuous products. 1282 and 1284) have minimum assay values of <95%.Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. 1271) will oxidize to a common metabolite. Consideration of secondary components Four members of this group of flavouring agents (Nos. unsaturated. Consideration of combined intakes from use as flavouring agents Seven (Nos 1272. 1274–1279) of the 20 substances are esters that will undergo hydrolysis to form cis-3-hexenol. In the unlikely event that all 20 agents considered here and the 42 agents considered previously were to be consumed concurrently on a daily basis. 3-hexenoic acid. Information on the safety of the secondary components of these four compounds is summarized in Annex 4 (Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less). The Committee concluded that under conditions of use the combined intake of these eight substances would not saturate the metabolic pathways leading to the common metabolite. acids and related esters (Nos 1269–1288). Evaluation of all the data indicated no safety concern associated with combined intake. trans-2-hexenal was considered not to present a 110 .

safety concern. One of the secondary components of No. 1279 (cis-3hexenyl-cis-3-hexenoate) was evaluated at the fifty-first meeting, and was considered not to present a safety concern at current levels of intake. The remaining secondary components of No. 1279 (isomers 1, 2, and 3 of hexenyl hexenoate) are expected to undergo the same metabolic pathway as 3-hexenyl-3-hexenoate, which includes hydrolysis to hexenol and hexenoic acid; 3-hexen-1-ol and 3-hexenoic acid were evaluated at the fifty-first meeting. On this basis, the secondary components of No. 1279 were considered not to present a safety concern at current levels of intake. The secondary component of No. 1282 ((E)-5-octenyl propionate) is expected to undergo the same metabolic pathway as the primary compound, which includes hydrolysis to cis-5-octen-1-ol and propionic acid; both of which were evaluated previously at the forty-ninth and fifty-first meetings, and considered not to present a safety concern. Although the secondary component of No. 1284 ((E,E)-3,6-nonadien-1-ol) has not been evaluated previously, a structurally related compound ((E,E)-2,6dodecadienal) was evaluated at the present meeting, and was considered not to present a safety concern based on current intake levels.
Conclusions

The Committee concluded that these 20 flavouring agents, which are additions to the group of linear and branched-chain aliphatic unsaturated primary alcohols and non-conjugated aldehydes, acids, and related esters evaluated previously, would not pose a safety concern at the currently estimated levels of intake. A monograph was published previously (Annex 1, reference 137).
4.1.7 Simple aliphatic and aromatic sulfides and thiols:

additional compounds

The Committee evaluated 12 flavouring agents that included simple aliphatic and aromatic sulfides and thiols (see Table 9) by the Procedure for the Safety Evaluation of Flavouring Agents (see Fig. 1). At its fifty-third meeting, the Committee had evaluated 137 other members of this chemical group of flavouring agents (Annex 1, reference 143). The group was divided into 12 subgroups on the basis of the position of the sulfur atom in order to facilitate the assessment of the relevant data on metabolism and toxicity. All 137 substances in that group were concluded to be of no safety concern on the basis of currently estimated levels of intake. Of the 12 additional flavouring agents considered here, six agents are thiols with oxidized side chains (subgroup v) (Nos 1289–1294) and
111

112

Table 9 Summary of results of the safety evaluations of simple aliphatic and aromatic sulfides and thiolsa,b,c CAS No. and structure Step B3 d Does intake exceed the threshold for human intake?

Flavouring agent Comments on predicted metabolism

No.

Step B4 Adequate margin of safety for substances or related substances?

Conclusion based on current intake

Subgroup ii — Acyclic sulfides with oxidized side chains Structural class I 2-(Methylthio)ethanol 1297 5271-38-5 No Europe: 1 S OH USA: 0.9

No safety concern

1298 233665-98-0
O S O

Ethyl 5(methylthio)valerate No Europe: 2 USA: 2

Yes. The NOEL of 1.4 mg/kg of See notes 7 body weight per day for the and 2 related substance 2(methylthiomethyl)-3phenylpropenal (No. 505) is >10 000 times the estimated daily intake of 2(methylthio)ethanol when used as a flavouring agent Yes. The NOEL of 1.4 mg/kg of See notes 5 body weight per day for the and 7 related substance 2(methylthiomethyl)-3phenylpropenal (No. 505) is >10 000 times the estimated daily intake of ethyl 5(methylthio)valerate when used as a flavouring agent

No safety concern

Subgroup iii — Cyclic sulfides Structural class III spiro(2,4-Dithia-11296 38325-25-6 methyl-8oxabicyclo(3.3.0)octaneS 3,3¢-(1¢-oxa-2¢-methyl)S cyclopentane)
O

No Europe: ND USA: 2 See notes 10, 1 and 3

No safety concern

O

Yes. The NOEL of 25 mg/kg of body weight per day for spiro (2,4-dithia-1-methyl-8oxabicyclo(3.3.0)octane-3,3’(1’-oxa-2’-methyl)cyclopentane) is >100 000 times the estimated daily intake when used as a flavouring agent

Subgroup v — Thiols with oxidized side chains Structural class I erythro- and threo-31289 Mercapto-2SH methylbutanol No Europe: 1 USA: 2
OH

See notes 1 and 2

No safety concern

(±)2-Mercapto-2methylpentan-1-ol
HS

1290 258823-39-1 No Europe: 3 USA: 4

See notes 1 and 2

No safety concern

OH

Yes. The NOEL of 0.7 mg/kg of body weight per day for the related substance 2mercapto-3-butanol (No. 546) is >10 000 times the estimated daily intake of erythro- and threo-mercapto2-methylbutan-1-ol when used as a flavouring agent Yes. The NOEL of 0.7 mg/kg of body weight per day for the related substance 2mercapto-3-butanol (No. 546) is >10 000 times the estimated daily intake of (±)2-mercapto-2methylpentan-1-ol when used as a flavouring agent

113

01 USA: 0. 546) is >10 000 times the estimated daily intake of 3-mercapto-2-methylpentenal when used as a flavouring agent Yes.02 Yes.7 3-Mercapto-2methylpentanal H SH O 1292 227456-28-2 No Europe: 3 USA: 4 See notes 1 and 4 No safety concern 4-Mercapto-4-methyl-2pentanone SH O 1293 19872-52-7 No Europe: 0. The NOEL of 0. 560) is >10 000 times the estimated daily intake of 4-mercapto-4-methyl-2pentanone when used as a flavouring agent See notes 1 and 3 No safety concern .9 mg/kg of body weight per day for the related substance 3mercapto-2-pentanone (No.7 mg/kg of body weight per day for the related substance 2mercapto-3-butanol (No.Table 9 (continued) CAS No. and structure Step B3 d Does intake exceed the threshold for human intake? 114 Flavouring agent Comments on predicted metabolism Conclusion based on current intake No safety concern See notes 1 and 2 No. Step B4 Adequate margin of safety for substances or related substances? 3-Mercapto-2methylpentan-1ol (racemic) OH SH 1291 227456-27-1 No Europe: 1 USA: 0. The NOEL of 0.7 mg/kg of body weight per day for the related substance 2mercapto-3-butanol (No. The NOEL of 1. 546) is >10 000 times the estimated daily intake of 3-mercapto-2-methylpentan1-ol (racemic) when used as a flavouring agent Yes.

trithiane (No.5-Trithiahexane 1299 42474-44-2 S S S No Europe: 0.3 mg/kg of body weight per day for the related substance 3-methyl1.04 Yes. The NOEL of 0.4.7 mg/kg bw per day for the related substance 2-mercapto-3butanol (No.3. 546) is >10 000 times the estimated daily intake of (±)ethyl 3mercaptobutyrate when used as a flavouring agent See notes 1 and 5 No safety concern Subgroup vii — Simple disulfides Structural class I 2.8 mg/kg of body weight per day for the related substance dipropyltrisulfide (No.006 USA: 0.2. The NOEL of 4. 574) is >10 000 times the estimated daily intake of 2. 8 and 9 No safety concern 115 . 8 and 9 No safety concern Subgroup ix — Trisulfides and polysulfides Structural class I Diisopropyl trisulfide 1300 5943-34-0 S S S No Europe: 0. 585) is >100 000 times the estimated daily intake of diisopropyl trisulfide when used as a flavouring agent See notes 7.5trithiahexane when used as a flavouring agent See notes 7.3.007 Yes.(±)Ethyl 3mercaptobutyrate O SH O 1294 156472-94-5 No Europe: 4 USA: 4 Yes. The NOEL of 0.03 USA: 0.

The ketone group is expected to be reduced to the alcohol. 10. and structure Step B3d Does intake exceed the threshold for human intake? 116 Flavouring agent Comments on predicted metabolism Conclusion based on current intake No. respectively. conjugated and subsequently excreted. Thioketal will hydrolyse to liberate the corresponding ketone and dithiol. The hydroxy group is expected to undergo oxidation to the carboxylic acid.5 mg/kg of body weight per day for the related substance ethylthioacetate (No. 2. Sulfur is expected to be oxidized to sulfonic acid. respectively. The combined per capita intake of the remaining flavouring agent in structural class III is 2 mg per day in the USA. The thioester is expected to undergo hydrolysis to acetate and the corresponding thiol. 7. 5 and 6 No safety concern CAS: Chemical Abstracts Service. Notes to Table 9: 1. the group was divided into 12 subgroups based on the position of the sulfur atom. conjugated and subsequently excreted. The subgroup designations are indicated in the table. 483) is >10 000 times the estimated daily intake of ethyl 4(acetylthio)butyrate when used as a flavouring agent See notes 1.Table 9 (continued) CAS No. 8. 4. d The threshold for human intake for structural class I. Step B4 Adequate margin of safety for substances or related substances? Subgroup xi — Thioesters Structural class I Ethyl 1295 104228-51-5 O 4-(acetylthio)butyrate O O S No Europe: 4 USA: 4 Yes. 1296) is in structural class III. undergo alkylation and conjugation followed by excretion. b Step 1: Eleven flavouring agents are in structural class I and one (No. 6. 540 and 90 mg/day. c Step 2: All of the agents in this group cannot be predicted to be metabolized to innocuous products. II and III are 1800. The combined per capita intake of the 11 flavouring agents in structure class I is approximately 21 mg per day in Europe and 24 mg per day in the USA. The ester is expected to undergo hydrolysis to the corresponding carboxylic acid and alcohol. The aldehyde group is expected to be oxidized to the corresponding carboxylic acid. Free thiols may form mixed disulfides with glutathione or cysteine. The NOEL of 6. The di. 3. 5. All intake values are expressed in mg/day. The cumulative per capita intake for the amended group as a whole including the 137 substances in the original evaluation and the 12 additional substances is 1181 and 1034 mg/person per day in Europe and the USA. which will be further oxidized. . To facilitate the evaluations. 9. The sulfur is expected to be oxidized to the sulfoxide and sulfone.or trisulfides are expected to be reduced to free thiols. ND: no data on intake reported a One hundred and thirty-seven (137) flavouring agents in this group were previously evaluated by JECFA.

Acyclic sulfides with oxidized side-chains (Nos 1297. The daily per capita intake of each agent is reported in Table 9. undergo methylation to yield methyl sulfides which then form sulfoxides and sulfones.contain an additional alcohol. cauliflower. wine. Absorption. Estimated daily per capita intake The total annual volume of production of the 12 simple aliphatic and aromatic sulfides and thiols is approximately 150 kg in Europe and in the USA. metabolism and elimination All of the sulfur-containing flavouring agents considered in this addendum are of low relative molecular mass and are sufficiently lipophilic to be absorbed. are conjugated with glucuronic acid. aldehyde. Two agents are acyclic sulfides with oxidized side-chains (subgroup ii) (Nos 1297 and 1298) in which an alcohol or ester functional group is present. a trisulfide (subgroup ix) (No. such as an alcohol (No. hop oil. Seven of the 12 flavouring agents in this group are naturally occurring components of food (Nos 1291–1294. react with endogenous thiols to form mixed disulfides. a disulfide (subgroup vii) (No. cabbage. reference 143). 1295). Metabolic options for simple thiols include oxidation to form unstable sulfenic acids (RSOH) which are oxidized to sulfinic acids (RSO2H). broccoli. 1300) and a cyclic sulfide (subgroup iii) (No. Thiols with oxidized side-chains (Nos 1289–1294) The metabolism of thiols with oxidized side-chains is predicted to involve a combination of pathways for simple thiols together with further oxidation or conjugation of the oxidized side-chain. 1296). The biotransformation of such oxygenated groups is well characterized and has been described for groups of flavouring agents evaluated 117 . 1300) and have been detected in onions. 1298). 1299). 1297) or ester (No. or undergo oxidation of the a-carbon which results in desulfuration and the formation of an aldehyde. or ester functional group. 1299. distribution. 1297. fish and cheese. ketone. None of these agents has been evaluated previously. 1298) The presence of oxygenated functional groups. and the presence of these polar sites would result in increased renal excretion of these agents. The remaining four substances are a thioester (subgroup xi) (No. fruits. These flavouring agents can be expected to be metabolized through the various pathways described below and in the previous evaluation by the Committee (Annex 1. provides additional sites for biotransformation of sulfides (thioethers).

Step 2. Application of the Procedure for the Safety Evaluation of Flavouring Agents Step 1. references 131. The remaining flavouring agent (No. 1300) The trisulfide of glutathione is labile and readily converted to the disulfide. by glutathione reductase or thioltransferases. by exchange with glutathione. thioredoxin. which are metabolized via the various pathways described above for simple thiols. At currently estimated levels of intake. Sulfoxide formation usually predominates as the major metabolic detoxication pathway. Trisulfides (No. 1295) Thioesters are hydrolysed by lipase and esterases. as well as chemically. Simultaneous metabolism of sulfur and oxygenated functional groups has been reported for various substrates. 138. 1296) was assigned to class III. Reduction of noncyclic disulfides (No. 132. the rate of hydrolysis increases as the length of the carbon chain increases and decreases as the oxygenation of the carbon chain in the thiol moiety increases. After hydrolysis. the resulting alcohol and carboxylic acid would participate in the metabolic pathways described above for sulfides containing oxygenated functional groups. The evaluation of these substances therefore proceeded via the B-side of the decision-tree. 144). Thioesters (No. the Committee assigned 11 agents (Nos 1289–1295. 118 . Trisulfides are predicted to be converted rapidly to the corresponding disulfides with subsequent reduction to thiols. with the release of sulfur as hydrogen sulfide. In applying the Procedure for the Safety Evaluation of Flavouring Agents to these 12 flavouring agents. 1296) Cyclic sulfides can be expected to undergo extensive S-oxidation by the cytochrome P450 superfamily to produce the corresponding sulfoxides. cysteine or other endogenous thiols. Cyclic sulfides (No. which would then be metabolized via the various pathways described above for simple thiols. 1299) The reduction of xenobiotic disulfides is believed to be extensive and can be catalysed enzymatically. none of the flavouring agents in this group is predicted to be metabolized to innocuous products. 1297–1300) to structural class I. Simple disulfides (No. 1299) would result in the formation of thiols of low molecular mass.previously by the Committee (Annex 1.

1291). 1800 mg).and threo-3-mercapto-2-methylbutanol (No.5-trithiahexane (No. 1299). 1298) which is also an acyclic sulfide with an oxidized side-chain that is anticipated to undergo oxidation and subsequent metabolism via similar pathways. 1297). 483) provides an adequate margin of safety (>10 000) in relation to known levels of intake of this agent. 574) provides an adequate margin of safety (>10 000) in relation to known levels of intake of this agent. For 2. the evaluation of all 12 agents in the group proceeded to step B4. 1293). For 4-mercapto-4-methyl-2-pentanone (No. 1289). For erythro. because they are all acyclic thiols with oxidized side-chains that are anticipated to undergo oxidation or hydrolysis and subsequent metabolism via similar metabolic pathways. 90 mg).3.2.4-trithiane (No.7 mg/kg body weight per day for the structurally related substance 2-mercapto-3-butanol (No. the NOEL of 0. 1292). 505) provides an adequate margin of safety (>10 000) in relation to known levels of intake of this agent. 1290).e. For ethyl 2-(methylthio)ethanol (No. The estimated daily per capita intakes of the 11 flavouring agents in this group in structural class I are below the threshold of concern (i. and (±)-ethyl 3-mercaptobutyrate (No. The estimated daily per capita intake for the one flavouring agent in structural class III is below the threshold of concern (i. 1294). This NOEL is also appropriate for the structurally related agents (±)-2-mercapto-2-methylpentan-1-ol (No. the NOEL of 1. Accordingly. 560) administered to rats by gavage in a 92-day study provides an adequate margin of safety (>10 000) in relation to known levels of intake of this agent. 3-mercapto-2methylpentan-1-ol (racemic) (No. 119 . the NOEL of 0. the NOEL of 1.3 mg/kg of body weight per day reported in a 13-week study in rats fed with the structurally related substance 3-methyl-1.5 mg/kg of body weight per day reported in a 13-week study in rats fed with the structurally related substance ethylthioacetate (No. Step B4. This NOEL is also appropriate for the structurally related agent ethyl 5(methylthio)valerate (No. the NOEL of 6.Step B3. For ethyl 4-(acetylthio)butyrate (1295).9 mg/kg of body weight per day for the structurally related substance 3-mercapto-2-pentanone (No.4 mg/kg of body weight per day reported in a 13-week study in rats fed by gavage with the structurally related substance 2-(methylthiomethyl)3-phenylpropenal (No.e. 3-mercapto-2methylpentanal (No. 546) from a 92-day study in rats fed by gavage provides an adequate margin of safety (>10 000) in relation to known levels of intake of this agent.

Consideration of secondary components One member of this group of flavouring agents (No. would not give rise to safety concerns at the currently estimated levels of intake. 1293. For spiro(2. 1300). In the unlikely event that the one agent considered in this evaluation and the six agents considered previously in structural class III were to be consumed concurrently on a daily basis. the NOEL of 25 mg/kg of body weight per day in the diet reported in a 13-week study in rats provides an adequate margin of safety (>100 000) in relation to known levels of intake of this agent. the estimated combined intake would not exceed the daily per capita human intake threshold for class I (1800 mg). Conclusion The Committee concluded that these 12 flavouring agents. 1296).8 mg/kg of body weight per day reported in a 13-week study in rats fed by gavage with the structurally related substance dipropyltrisulfide (No. the NOEL of 4.For diisopropyl trisulfide (No.0)octane-3.3. The secondary component (4-methyl-3penten-2-one) was evaluated at the fifty-ninth meeting. 4mercapto-4-methyl-2-pentanone) has a minimum assay value of <95%. reference 143) summarizing the safety data on these 12 additional members was prepared.3¢-(1¢-oxa2¢-methyl)-cyclopentane) (No. An addendum to the monograph for this group (Annex 1.4-dithia-1-methyl-8-oxabicyclo(3. which are additions to the group of simple aliphatic and aromatic sulfides and thiols evaluated previously. Table 9 summarizes the evaluations of the 12 simple aliphatic and aromatic sulfides and thiols in this group Consideration of combined intakes from use as flavouring agents In the unlikely event that the 11 agents considered in this evaluation and the 97 agents considered previously in structural class I were to be consumed concurrently on a daily basis. the estimated combined daily per capita intake would not exceed the human intake threshold for class III (90 mg). 585) provides an adequate margin of safety (>100 000) in relation to known levels of intake of this agent. 120 . and was considered not to present a safety concern at current levels of intake. Information on the safety of the secondary component of this compound is summarized in Annex 4 (Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less).

4-hexadienoic acid (No. 605. pending receipt of more data. 615. the Committee noted that the existing specifications need to be updated. the Committee decided that revised specifications in the flavouring agents section should supersede those for food additives.2 Revision of existing specifications for flavouring agents The existing specifications for 101 flavouring agents were reviewed.2) were designated “tentative”. 399. tentative specifications. the Committee has agreed that the material used for flavouring should comply with the existing food additive specifications. 1225. 5. 631. 1291 and 1296) were designated “tentative”. six of which (Nos 1203. 1172. 68.1 Nutritional source of iron Ferrous glycinate (processed with citric acid) The call for data for the sixty-first meeting referred to this substance as ferrous bisglycinate. The substances in question are: benzyl butyl ether.2 and 633. 1176. citral.2 Revision of certain specifications for purity of flavouring agents the sixty-first meeting 4. dibenzyl ether. 504. It is therefore unnecessary to maintain separate flavourings specifications. 570. of which 14 (Nos 53. 1218. 1253 and 1256).2.4dihydrocoumarin and 6-methylcoumarin. 1273. Specifications in the flavouring agents format were prepared for the six remaining substances (Nos 1171. The flavouring agent (E. also known as sorbic acid) was evaluated previously as a food preservative and a specification was published. along with those for sorbate salts. 1263.2. 5. Revised specifications were adopted for all of these. Until this can be done.1 Specifications for flavouring agents evaluated for the first time at At its present meeting. 628.2. 4.E)-2. The Committee decided that this name did not 121 .4. 3. 55. the Committee reviewed 144 substances submitted for evaluation. citronellol. Since these substances have no other functional uses than as flavouring agents. 557. pending receipt of further data. 471. the Committee decided to maintain separate. New specifications were proposed for 139 flavouring agents. and withdrew the food additive specifications. In the case of sorbic acid. 1219. For such substances. 632. all of which have existing food additive specifications. The Committee noted that specifications for other food additives which are used as flavouring agents in addition to other uses may also need revision at future meetings.

references 107 and 143) and is generally greater than that of ferrous sulphate. the nature of the food matrix may affect the bioavailability of the iron from ferrous glycinate. As is the case with other non-haem iron compounds. except for iron oxides used as food colouring agents.8 mg/kg of body weight for iron from all sources. Annex 1. and is subsequently dissociated into its iron and glycine components within the intestinal mucosa. The resulting product is spraydried without prior removal of the citric acid. iron absorption from ferrous glycinate is down-regulated according to iron status. At its present meeting. The bioavailability of iron from ferrous glycinate is comparable to that of iron–EDTA (evaluated by the Committee at its forty-first and fifty-third meetings. reference 62). >97% of the ferrous ions are chelated. Ferrous glycinate (processed with citric acid) is an iron (II) chelate with the amino acid glycine. in a manner similar to other non-haem iron compounds. and supplemental iron for specific clinical requirements. and also contains citric acid. At chemical equilibrium. The Committee therefore concluded that there was no evidence that the administration of iron in the form of 122 . The substance is highly hygroscopic and may contain water in variable amounts.accurately describe the substance being evaluated and therefore agreed that this substance should be referred to as ferrous glycinate (processed with citric acid). supplemental iron taken during pregnancy or lactation. the Committee allocated a provisional maximum tolerable daily intake of 0. Biological data. The available studies indicate that the absorption of iron from ferrous glycinate is regulated physiologically according to the body’s iron status. while showing no gastric side-effects. It is manufactured by the reaction of reduced iron with glycine. In consideration of the potential for overuse of this product. which confirmed the efficacy of ferrous glycinate in correcting iron status in individuals exhibiting iron deficiency. the Committee was asked to comment on the safety of ferrous glycinate as a source of iron for dietary supplementation and as a fortificant for general use in food products. the Committee noted the results of studies of dietary supplementation and fortification at doses of up to 60 mg iron per day. At its twenty-seventh meeting (Annex 1. The Committee noted that ferrous glycinate is absorbed by the mucosal cells of the intestine. and haemoglobin and serum ferritin concentrations are not significantly increased relative to pre-treatment or normal-range values at doses of up to 23 mg iron per day. in the presence of citric acid. In iron-sufficient individuals. including children.

Products which are intended to provide a source of additional iron. no compound-related toxicological effects at doses of 100. The Committee did not receive information concerning estimated intakes for ferrous glycinate.1 Disinfectant for drinking-water Sodium dichloroisocyanurate Sodium dichloroisocyanurate (NaDCC) is the sodium salt of a chlorinated hydroxytriazine and is used as a source of free available chlo123 . and toxicity. a chemical and technical assessment (CTA) and specifications were prepared. suggest that intakes approaching the provisional maximum tolerable daily intake could not be attained without consuming extremely large amounts of foodstuffs fortified at the suggested levels. 6. In preparing the specifications. except under medical supervision. should not be consumed by individuals with any type of iron storage disease.8 mg/kg of body weight for iron from all sources. The NOEL for this study was reported to be 500 mg ferrous glycinate/kg of body weight per day. A toxicological monograph. Evaluation. and the studies in humans. provided by the sponsor. Information on levels of fortification in food. and is usually formulated with diluents and flow agents to facilitate the manufacture of iron-fortified food products. corresponding to 100 mg iron/kg of body weight per day. provided that the total intake of iron did not exceed the provisional maximum tolerable daily intake of 0. 6. Despite the fact that a slight increase in iron deposition in the liver of rats of each sex occurred at high doses. 250 or 500 mg/kg of body weight per day were noted. the Committee was aware that food-grade ferrous glycinate (processed with citric acid) is commercially available.ferrous glycinate would result in increased body stores of iron after the nutritional requirement for iron had been satisfied.8 mg/kg of body weight. The Committee reviewed a 90-day study of toxicity in rats fed diets containing ferrous glycinate. On the basis of the available data on bioavailability. This NOEL is 125-fold the provisional maximum tolerable daily intake of 0. the Committee concluded that ferrous glycinate was suitable for use as a source of iron for supplementation and fortification. including ferrous glycinate. metabolism. either from its use in food or any possible use as an iron supplement.

the daily intake of the dissociation products of NaDCC from the consumption of water by adults. according to WHO estimates. WHO also recognizes that higher intake rates may occur in some tropical countries. 124 . and 0. As free available chlorine is consumed by reaction with organic material in the water. would be equivalent to 6. The default upper-percentile drinking-water intake rates currently used by WHO are 2 litres per day for adults. and 0. 1. 1 litre per day for a 10-kg child. chloroisocyanurates will rapidly dissociate and continue to release free chlorine. NaDCC can be manufactured either as the anhydrous salt or as the dihydrate.4. to overcome initial chlorine demand. A typical concentration of free available chlorine used for the treatment of drinking-water is 1. The use of NaDCC as a source of free available chlorine is not expected to lead to greater production of such by-products than does the use of elemental chlorine. The safety of these by-products has been addressed by WHO. Conventional chlorination of drinking-water with elemental chlorine gives rise to a number of by-products as a result of the reaction of free available chlorine with natural organic matter. assuming a maximum application of 3. 3. As anhydrous NaDCC contains about 63% free available chlorine.2 mg/ l).6 mg/l NaDCC (or 1. 0.06 mg/kg of body weight for adults. and 2. It has not been evaluated previously by the Committee. These intakes include water consumed in the form of juices and other beverages containing tap water (e. the Committee considered the safety of NaDCC in relation to its possible use as a disinfectant for drinking-water in emergency situations. ingestion of cyanuric acid is estimated to be 0.8 mg/l of the dihydrate) is equivalent to 1 mg/l free available chlorine. When NaDCC is added to water. and for routine use in some water supplies. it is rapidly hydrolysed to release free available chlorine.2 mg NaDCC per litre. However.e.75 litres per day for a 5-kg bottle-fed infant.19 mg/kg of body weight for children. expressed as NaDCC.2. At its present meeting. Drinkingwater becomes increasingly unpalatable as concentrations of free chlorine increase above this level.28 mg/kg of body weight for a bottlefed infant. HOCl) for the disinfection of drinking-water. 3. establishing a complex series of equilibria involving six chlorinated and four non-chlorinated isocyanurates. Given that 1 mole of NaDCC corresponds to 1 mole of cyanuric acid (the ultimate endproduct of the application of NaDCC). coffee). children and infants.rine (in the form of hypochlorous acid. Thus. respectively. disinfection using NaDCC might require higher initial doses.4 mg/person per day. but not greater than double these quantities (i. with the development of guidelines for drinkingwater quality.g.0 mg/l.

The relevant toxicological studies cited refer to this compound.In contact with saliva of about pH 7.5–2. Charles River CD-1 rats were given sodium cyanurate in the drinking-water at doses estimated as 26. Only 5% of the administered dose was detected in the faeces and the radiolabel was not exhaled as 14Ccarbon dioxide. There was no substance-related increase in tumour incidence. therefore. Both NaDCC and sodium cyanurate have low acute oral toxicity. the sodium cyanurate was extensively absorbed and excreted unchanged in the urine. In studies in which 14C-labelled sodium cyanurate was administered in multiple doses of 5 mg/kg of body weight to rats. the only compound-related effect reported was the occurrence of bladder calculi in males receiving the highest dose. or untreated drinking-water. no detectable chlorinated isocyanurate remains. Survival was slightly lower in the group receiving the highest dose compared to the control group receiving untreated drinking-water. between 2% and 13% of 14 C-labelled sodium cyanurate was excreted unchanged in the faeces and the remainder in the urine. 77. at the concentrations required to deliver free available chlorine at the levels typically used in drinking-water. No toxicologically significant treatment related effects 125 . dilated and inflamed ureters and renal tubular nephrosis) and cardiac lesions (acute myocarditis. Multiple lesions of the urinary tract (calculi and hyperplasia. In a 2-year study. In a similar study in Charles River rats. The elimination half-life was 40–60 minutes in the rat. but not the control group receiving sodium hippurate. The material that reaches the gastrointestinal tract is. mainly within about 6 hours. chlorinated isocyanurates react extremely rapidly such that. 1. the unchlorinated cyanuric acid. In two human volunteers given a solution of cyanuric acid of unspecified concentration.0. with control groups receiving drinking-water containing an equivalent amount of sodium hippurate. mainly within 12 hours. In 13-week studies in mice given up to 5375 mg/l of sodium cyanurate (equivalent to 1500 mg/kg of body weight per day) in drinking-water. bleeding and inflammation of the bladder epithelium. greater than 98% of the cyanurate was recovered unchanged in the urine after 24 hours. 154 or 371 mg/ kg of body weight.0 hours in the dog and about 3 hours in humans. 1 out of 28 males in the group receiving 1792 mg/ l (equivalent to 145 mg/kg of body weight per day) and 7 out of 28 males in the group receiving the highest dose (equivalent to 495 mg/kg of body weight per day) showed epithelial hyperplasia of the bladder. necrosis and vascular mineralization) were reported in males that died during the first year of the study and that were receiving a dose of 371 mg/kg. In a similar study in the dog.

The NOEL for sodium cyanurate derived from the 2-year study in rats was 154 mg/kg of body weight per day. with control groups receiving untreated drinking-water or sodium hippurate.0 mg anhydrous NaDCC/kg of body weight per day was determined by the Committee for intake from drinking-water treated with NaDCC for the purpose of disinfection. Three generations of Charles River CD rats were given doses estimated to be 26. equivalent to 220 mg anhydrous NaDCC/kg of body weight per day. was also observed in this strain in the group given a dose of 500 mg/kg. either Dutch belted or New Zealand White. because any residues of intact NaDCC in drinking-water would be rapidly converted to cyanuric acid on contact with saliva. An increased incidence of postimplantation loss. 110. F1 and F2 generations or on offspring of the F1. 200 or 500 mg/kg of body weight per day of sodium cyanurate was administered by gavage on days 6–18 of gestation. Sodium cyanurate did not induce any genotoxic.were observed at 154 mg/kg of body weight. a small reduction in body-weight gain was observed in the groups receiving the two highest doses on days 12–19 of gestation in New Zealand White rabbits only. 50. There were no signs of toxicity in adult animals and no effects reported in the offspring of groups of Charles River COB and CD rats given sodium cyanurate at doses of 0. The Committee concluded that studies of the toxicity of sodium cyanurate were appropriate for assessing the safety of sodium dichloroisocyanurate. In a similar 2-year study in which B6C3F1 mice received doses of sodium cyanurate equivalent to 30. in which 0. 340 or 1523 mg/kg of body weight per day. 1000 or 5000 mg/kg of body weight per day by gavage on days 6–15 of gestation. F2 or F3 generations . Sodium cyanurate was not genotoxic in four different tests. With the application of an uncertainty factor of 100. which was considered to be the NOEL in this study. but compensatory weight gains were made by the end of the study. In studies of pregnant rabbits. survival was similar in all groups and there were no treatment-related changes in the incidence of tumours or other histopathological lesions. 126 . a tolerable daily intake (TDI) of 0– 2. The Committee considered that these effects were not significant and there were no other effects that were considered to be related to treatment. There were no treatmentrelated effects on reproductive parameters in the P0. 77 or 100 mg/kg of body weight sodium cyanurate in their drinking-water. carcinogenic or teratogenic effects. 200. which was within the historical control range.

age dependency of the intake:excretion ratio). copper. 149). references 30. calcium. 7.g.1 Contaminants Cadmium Cadmium was evaluated by the Committee at its sixteenth. the oral bioavailability of cadmium ranged from 0. At its fifty-fifth meeting. the Committee allocated a provisional tolerable weekly intake (PTWI) of 400–500 mg of cadmium per person.A toxicological monograph and a chemical and technical assessment (CTA) were prepared and new specifications were established to cover both anhydrous NaDCC and the dihydrate. thirtythird. and nutritional status. The Committee made several recommendations regarding the data that would be needed in order to reduce the uncertainty in the prevalence estimates.2 Observations in animals In the experimental animal species tested. The Committee noted. and concluded that the risk of excess renal tubular dysfunction in the population would be negligible below a urinary cadmium excretion of 2. 83. and iron have been shown to promote cadmium absorption 127 . Low dietary concentrations of protein and of essential minerals such as zinc. 7. At its sixteenth meeting.5–3.5 mg/g of creatinine.0% on average. dietary bioavailability. At the three subsequent meetings. the Committee retained this PTWI. developmental stage. the Committee at the fiftyfifth meeting maintained the PTWI at this value because of lack of precision in the risk estimates.1. Experimental studies also identified various factors that can significantly influence the extent of cadmium absorption and retention from the diet. but expressed it in terms of the intake of cadmium per kg of body weight (7 mg/kg of body weight). including sex. A considerable number of new studies addressed certain aspects of the issues identified in these recommendations and served as the basis for the Committee’s deliberations at the present meeting. that these estimates are based on a model that is dependent on the values assumed for key parameters (e. however. 7.1. the Committee decided that the prevalences of renal tubular dysfunction that correspond to various dietary intakes of cadmium could serve as a reasonable basis for risk assessment. 107.1 Introduction 7. forty-first and fifty-fifth meetings (Annex 1. Although new information indicated that a proportion of the general population might be at an increased risk of tubular dysfunction at the current PTWI of 7 mg/kg of body weight.

including proximal tubule epithelial cell damage. After absorption. Long-term oral exposure to cadmium resulted in a variety of progressive histopathological changes in the kidney. high or adequate dietary concentrations reduce cadmium absorption and retention. A variety of immune system effects have been observed in experimental animals exposed to cadmium. developmental neurobehavioural effects. Investigations into the ability of cadmium compounds to induce developmental effects in experimental animals have shown that decreased fetal weight. Decreases in bone calcium concentrations and increased urinary excretion of calcium have also been associated with exposure to cadmium. Recent studies conducted in Japan. China. However. have been seen in the absence of any overt symptoms of maternal toxicity and appear to be a more sensitive indicator of toxicity. Biochemical indications of renal damage were seen in the form of low molecular weight proteinuria. glucosuria and aminoaciduria. 7. because of its long half-life in humans. and the United States have attempted to refine estimates of the dose–effect/dose– response relationship between environmental exposure to cadmium 128 . skeletal malformations and increased fetal mortality are common findings. including increased virusinduced mortality in mice co-exposed to non-lethal doses of cadmium and RNA viruses. Tubular dysfunction also caused the urinary excretion of cadmium to increase. Cadmium accumulates in the kidney and. particularly renal dysfunction. in contrast. mortality. Europe. Cadmium induced malignant transformation of animal and human cells in vitro. usually in combination with indices of maternal toxicity.while. cadmium is distributed mainly to the liver. interstitial fibrosis.3 Observations in humans A number of new epidemiological studies published since the fiftyfifth meeting have evaluated the relationship between exposure to cadmium and various health effects. and glomerular basal cell damage with limited tubular cell regeneration. including decreased locomotor and exploratory activity and certain electrophysiological changes. and calcium/bone metabolism.1. steady-state concentrations in the renal cortex are reached only after about 40 years. with subsequent redistribution to the kidney in conjugated forms such as cadmium–metallothionein and cadmium–albumin.

the prevalence of end-stage renal disease was found to be significantly.5–1 mg/g creatinine. individual biomarkers of exposure were not measured in this study. the new data are consistent with the hypothesis that low-level environmental exposure to cadmium is associated with an increased prevalence of renal proximal tubular dysfunction. as determined by area of residence. Simple adjustment for creatinine might be misleading if comparisons involve people differing in physique. physical activity. in one study.3 mg/g creatinine. Above a urinary cadmium concentration of 5 mg/ g creatinine. the long-term health implications of the changes in renal function observed at low concentrations of urinary cadmium are uncertain. although urinary b2microglobulin and N-acetyl-b-D-glucosaminidase (NAG) were measured as indices of tubular dysfunction in both studies. Two studies of populations with low concentrations of urinary cadmium (mean concentrations of 0. The appropriate concentrations of urinary biomarkers to use as cut-off values for identifying tubular proteinuria might also vary depending upon physiological or disease conditions.23 mg/g creatinine and 0. only b2-microglobulin was associated with urinary cadmium concentration while in the other study. However. although modestly. In an environmental study. sex. However. the prevalence of tubular proteinuria was increased five fold. In some studies. and race. the findings of these two studies were inconsistent. It is well-established that cadmium-induced low molecular weight proteinuria can progress to an acquired Fanconi syndrome (the continuous loss of calcium and phosphorus into urine) and/or the disturbance of vitamin D metabolism in the damaged kidneys. The latter 129 . only NAG was associated with urinary cadmium concentration. Finally. age. In a Swedish study (the OSCAR study) involving >1000 individuals aged 16–80 years.26 mg/g creatinine) found associations between markers of early kidney damage and urinary cadmium concentration. In aggregate. The epidemiological studies conducted in regions of Japan where levels of environmental cadmium vary identified several issues that complicate the interpretation of studies of renal function and low environmental exposure to cadmium. an increase of nearly threefold in the prevalence of tubular proteinuria was observed in the group with urinary cadmium concentrations of 0. related to the extent of environmental exposure to cadmium. a crude association between urinary cadmium and a biomarker of effect disappeared after adjusting for age.and renal dysfunction. compared to the group with a urinary cadmium concentration of <0. as assessed by biomarkers.

and only weakly with urinary cadmium concentration. the age. might alter calcium metabolism in bone tissue independently of renal effects. Bone metabolism is influenced by many factors. molluscs. and offal (especially liver and kidney). At its present meeting. which was due mainly to ageing. According to the results of the OSCAR study. This association was corroborated by the results of two earlier studies.and sex-adjusted risk of having a reduced bone mineral density was increased two-fold among individuals with blood-cadmium concentrations of 0. These estimates consti130 . after adjustment for age.7– 6.05 mg/kg in most foods. Mean dietary intakes derived from Global Environment Monitoring System — Food Contamination Monitoring and Assessment Programme (GEMS/Food) regional diets (average per capita food consumption based on food balance sheets) and average concentrations of cadmium in these regions range from 2. characterized by osteomalacia. although higher concentrations were found in nuts and oil seeds. although bone mineral density was correlated with age and body weight.6–1. but with deterioration of renal tubular function. and environmental factors such as sunlight. The combined data showed that concentrations of cadmium range from about 0. even at low concentrations. Japan.1. Croatia. body mass index. showed no correlation between exposure to cadmium and bone mineral density or calcium excretion. and might increase the risk of osteoporosis and bone demineralization. including age.1 mg/l and threefold among individuals with blood-cadmium concentrations >1. and the European Union. Greece.3 mg/kg of body weight per week. The excretion of calcium was not correlated with exposure to cadmium.01– 0.may eventually progress to Itai-itai disease. Two studies in Japan. estrogen status. physical activity. Spain. the Committee evaluated the dietary intake of cadmium using data from a number of countries. Estimates of the mean national intake of cadmium ranged from 0. Slovakia.8–4. Nigeria. 7. physique. ethnic group. These studies were therefore considered by the Committee to be preliminary.4 Estimated dietary intake At its fifty-fifth meeting. and menstrual status. one in Belgium and one in Japan.2 mg/kg of body weight per week. None of the studies adjusted for possible confounding by all of these factors.1 mg/l. Some recent reports suggest that environmental exposure to cadmium. the Committee updated its review by adding new information from Australia. one in which environmental exposure to cadmium was moderate and one in which it was high. France. Lithuania. nutritional status.

fetal growth. and neurotoxicity. wheat.1. The Committee reaffirmed its conclusion that an excess prevalence of renal tubular dysfunction would not be expected to occur if urinary cadmium concentration remains <2. the Committee noted inconsistencies between studies regarding the specific biomarkers of renal function that were most commonly associated with urinary cadmium concentrations. the Committee noted that appreciable uncertainty remains regarding the long-term health significance of these changes. Europe and the USA indicated that changes in renal function and bone/calcium metabolism are observed at urinary cadmium concentrations of <2. even under a range of plausible assumptions about the relationship between the amount of bioavailable cadmium in the diet and the urinary excretion of cadmium. starchy roots/tubers. the following foods contributed 10% or more to the PTWI in at least one of the GEMS/Food regions: rice. Although recent studies suggested that increased concentrations of cadmium biomarkers are associated with health effects such as diabetes.5 mg/g creatinine. Uncertainty remains about how these assumptions affect the predicted excess prevalence of renal tubular dysfunction at concentrations of urinary cadmium of >2. the total intake of cadmium might exceed the PTWI because total food consumption for high consumers is estimated to be about twice the mean. sufficiently robust to serve as a basis for the evaluation. that addressed issues identified as research needs at its fiftyfifth meeting.5 mg/g creatinine. In addition. pancreatic cancer. and therefore maintained the current PTWI of 7 mg/kg body weight.5 Evaluation The Committee considered an extensive amount of new information. Although the sensitive biomarkers used by some recent studies conducted in Japan. Regarding the major dietary sources of cadmium.5 mg/g creatinine. The Committee reaffirmed its conclusion that renal tubular dysfunction is the critical health outcome with regard to the toxicity of cadmium. hypertension. 7. particularly from a series of Japanese environmental epidemiological studies. For some individuals. No excess prevalence of renal tubular dysfunction would be predicted to occur at the current PTWI under the most appropriate assumptions about the fractional bioavailability of cadmium and the percentage of the absorbed cad131 . at this time.tute approximately 40–60% of the current PTWI of 7 mg/kg of body weight. and molluscs. The Committee concluded that the new data which became available since its fifty-fifth meeting do not provide a sufficient basis for revising the PTWI. the Committee concluded that these data were not. Vegetables (excluding leafy vegetables) contribute >5% to the PTWI in two regions.

references 30. the Committee reviewed many experimental results which indicated that the developing nervous 132 . the Committee reaffirmed the previously established provisional tolerable weekly intake (PTWI) of 200 mg methylmercury (3. twenty-second. but noted that fetuses and infants might be more sensitive than adults to its toxic effects. especially in certain regions.2 Observations in animals In its previous assessment.2 Methylmercury 7.1 Introduction Methylmercury was evaluated by the Committee at its sixteenth. different methods for assessing neurobehavioural effects had been used in the two cohorts. The Committee recommended that methylmercury be reevaluated at a subsequent meeting when the results of the analysis of neurodevelopmental effects in the Seychelles cohort after 8 years and other relevant data had become available. which were evaluated at its fifty-third meeting. Adverse effects on neurodevelopment were reported in the study in the Faroe Islands. 7. did not provide consistent evidence concerning neurodevelopmental effects in children of women whose methylmercury intakes had resulted in hairmercury burdens of 20 mg/kg of body weight and below. 47. The Committee noted that fish make an important nutritional contribution to the diet. but not in that in the Seychelles. PTMI) for contaminants with longer biological half-lives. At the last meeting. 83. 7. The Committee recommended that the evaluation of cadmium should be revisited when this project has been completed. 144). nutritional benefits should be weighed against the possibility of adverse effects.2.g. Studies published since the fifty-third meeting were considered at the present meeting.mium that is excreted in urine.3 mg/kg of body weight) for the general population. The Committee noted that two issues being considered by the Joint FAO/WHO Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food were of particular relevance to the present evaluation: the dose– response assessment of biomarkers of effect and their relationship to disease outcome. and recommended that nutritional benefits be weighed against the possibility of adverse effects when limits were being considered for methylmercury concentrations in fish or for fish consumption. The Committee concluded that data from studies undertaken in the Seychelles and the Faroe Islands.2. and the possible specification of longer tolerable intake periods (e. thirty-third and fifty-third meetings (Annex 1. however.

7. the indices of neurotoxicity include neuronal loss. but the results are conflicting. kidney. and inhibition of axonal morphogenesis and cell cycle progression. induction of reactive oxygen species and oxidative DNA damage. decreasing spleen and thymus cell viability. and reduced offspring viability. ataxia. The neurotoxic effects observed in non-human primates were consistent with the symptoms of Minamata disease. increased the frequency of fetal resorption and malformations. loss of coordination. methylmercury was readily absorbed (up to 95%) after oral exposure. Exposure of neuroepithelial cells to methylmercury in vitro resulted in disruption of intracellular calcium homeostasis. In all experimental animal species evaluated.system. decreased activity.2. Both the central and peripheral nervous systems show signs of damage. Ataxia. is a sensitive target for methylmercury. visual disturbances. Methylmercury is eliminated mainly via the bile and faeces. Methylmercury also affected the rodent immune system. the Committee confirmed that neurotoxicity is the most sensitive endpoint. In rodents. In humans. reducing mast cell function and. resulting in higher concentrations of mercury in the brain of the fetus than of the mother. impaired hearing. Information about the neurotoxicity caused by chronic fetal exposure to low doses of methylmercury has come primarily from epidemio133 . Changes in behaviour. liver and reproductive organs. and developmental stage. the Committee noted that methylmercury is toxic to the nervous system. At its present meeting. neonatal animals having a lower excretory capacity than adults. paralysis and death. The nature and severity of symptoms depend on dose and duration of exposure.3 Observations in humans At its fifty-third meeting. neurotoxic effects attributable to methylmercury usually become evident at doses that also affect other organ systems. particularly in non-human primates. Experimental evidence indicates a possible protective effect of selenium against some toxic effects of methylmercury. and deficiencies in learning and memory have also been observed. at high oral doses. the syndrome observed in humans poisoned with methylmercury via consumption of contaminated seafood. Methylmercury crossed both the blood–brain barrier and the placenta effectively. Treatment of pregnant female rodents with methylmercury induced abortion. paralysis. and hind limb crossing are common neurological signs of exposure to methylmercury in rodents.

Additional analyses of the assessments of the cohort at 7 years of age were carried out to explore the possibility of age. although these were cross-sectional rather than prospective in design. A few new epidemiological studies of neurodevelopment have been reported. and involved much smaller sample sizes than either the Seychelles or Faroe Islands studies. no adverse effects have been detected that are attributable to prenatal exposure to methylmercury. and more detailed evaluation of specific test scores. The results did not support a role for any of these factors in the positive associations observed in this study. Analyses were also conducted to determine whether the methylmercury-associated neuropsychological deficits observed in this cohort were attributable to episodes of higher exposure to methylmercury during pregnancy (associated with consumption of whale-meat). and provide no evidence for an inverse association between maternal exposure to methylmercury and neurodevelopmental effects in the children. No new data were available from the main Faroe Islands study. Further analyses of the results of assessments of the Seychellois children at the age of 5. exposure to higher concentrations of methylmercury.5 years have been published. and effects on children’s visual function. adjustment for additional potential confounding factors. A cross-sectional study of neurotoxic effects in adults reported significant mercury-associated neurobehavioural deficits in persons whose current hair-mercury 134 . Many of the neuropsychological test instruments included in the battery were the same as those used in the study in the Faroe Islands and which had been observed to be associated with biomarkers of prenatal exposure to methylmercury in 7-year-old children. prenatal exposure to methylmercury was found to be inversely related to newborn neurological status and to postnatal growth at 18 months of age. these present alternative statistical approaches. The results of these analyses do not alter the conclusion that in these populations with frequent fish consumption. The results of the neurodevelopmental assessments of 8-year-old children in the Seychelles study cohort were consistent with those obtained in this cohort previously. residual confounding due to concomitant exposure to polychlorinated biphenyls (PCBs). and. In a second smaller cohort (182 infants) assembled in the Faroe Islands. in most cases. The association was still present after adjustment for exposure to 28 PCB congeners and 18 organochlorine pesticides or their metabolites.and test-dependent variation on susceptibility to methylmercury.logical studies of populations in which fish consumption is frequent.

In a case– control study.2. although the frequencies of many neurological and neuromuscular symptoms were higher. over a 4-year follow-up interval. A number of dose–response assessments have been conducted using the results of the three major epidemiological studies of fetal neurotoxicity. hair-mercury concentrations of ≥2 mg/kg were associated with a doubling of the risk of suffering an acute myocardial infarction and. With regard to reproductive toxicity.4 Dose-response assessments The Committee concluded that neurotoxic effects resulting from exposure to methylmercury in utero were the most sensitive health outcome. whereas the other reported similar concentrations in the two groups. cardiotoxicity. and diabetes mellitus were not significantly increased in persons living near Minamata Bay. higher blood mercury concentrations were found in infertile than fertile couples. 135 . renal disease. The results of two large case–control studies of mercury exposure and coronary heart disease were conflicting. the Seychelles. suggesting that much of their exposure had been to metallic mercury rather than to methylmercury. the Committee considered that these results could not form the basis of a dose–response assessment. half the participants were dentists and had concentrations of toenail-mercury that were twice as high as those of non-dentists. With respect to cardiotoxicity. These assessments were made on the basis of evaluations of children at 7 years of age in the Faroes Islands study. a methylmercuryassociated decrease in the ratio of male : female births in the area of Minamata City during the period of peak pollution was reported. The Committee determined that the available evidence for the potential cardiotoxicity of methylmercury was not conclusive. high fish consumption. with an increased risk for atherosclerotic disease. In another study. In the latter study. and New Zealand. but noted that further studies were needed. the primary route of exposure to methylmercury. With regard to general health status. in a cohort study.concentration was <15 mg/kg. Additional epidemiological studies have addressed issues such as reproductive toxicity. but no biomarkers of mercury exposure were measured. conducted in the Faroes Islands. 7. one study reporting significantly higher concentrations of mercury in the toenails of cases than of controls. the prevalence rates of liver disease. immunotoxicity. but the ratio subsequently returned to control levels. and general medical status. Because of the cross-sectional design of this study and because an adult’s hair-mercury concentration does not accurately reflect concentrations during the critical exposure period for neurodevelopment. was associated with an increased risk of stroke.

The Committee noted.5. The concentration of methylmercury in maternal blood that would be expected to have no 136 . and a mathematical analysis of the concentration– response relationship was used to determine a benchmark-dose lower-confidence limit (BMDL) for the studies in the Faroes Islands and New Zealand. The Committee used the average from the two studies. 14 mg/kg maternal hair-mercury. however. the Committee confirmed the validity of these biomarkers for both short-term (blood) and longer-term (hair) intake of methylmercury. that the inclusion of the results of the study in New Zealand did not materially alter its evaluation. After consideration of numerous publications. more than four times the next highest concentration in the study sample and had a heavy impact on the BMDLs. The maternal hair-mercury concentration corresponding to a NOEL for neurobehavioural effects was identified for the study in the Seychelles. The Committee noted that the maternal hairmercury concentration of one child (out of 237) in the study in New Zealand was 86 mg/kg. Because of uncertainty about which set of BMDLs was most valid. Calculation of steady-state ingestion of methylmercury (mg/kg of body weight per day) from a maternal hair-mercury concentration comprises two steps: conversion of the concentration of methylmercury in maternal hair to that in maternal blood. A comprehensive dose–response assessment on the basis of the evaluations of the children in the Seychelles study at 8 years of age has not yet been reported. The mean ratio of the concentrations of methylmercury in hair to those in blood was determined in a number of studies. but the study results were similar to those obtained at 5. using samples from various study groups and with a variety of analytical methods.5 years of age. Mercury in maternal hair and/or cord blood served as the primary biomarkers of exposure to methylmercury in utero in the studies in the Faroe Islands and the Seychelles.4–10 mg/kg. the Committee decided to base the evaluation only on the results of the studies in the Faroe Islands and the Seychelles (see Table 10). as an estimate of the concentration of methylmercury in maternal hair that reflects exposures that would have no appreciable adverse effect on the offspring in these two study populations.5 years of age in the Seychelles Islands study. The inclusion of this observation produced BMDLs of 17–24 mg/kg. and 6 years of age in the New Zealand study. while omitting it produced BMDLs of 7. and conversion of the concentration of mercury in maternal blood into maternal intake. The Committee used a value of 250 to represent the overall average ratio. and was usually in the range of 140–370.

(1999. In humans.95) f = the absorbed fraction distributed to the blood (0. 2001).014 per day-1) V = blood volume (9% of body weight for a pregnant female) A = fraction of the dose absorbed (0. the maternal body burden at term is determined largely by intakes during the second and third trimesters of pregnancy.3 14.5 mg/kg of body weight per day would result in a maternal blood-mercury concentration that would 137 . Using this equation. (2003) US ATSDR (1999) Not applicable appreciable adverse effects on the offspring was calculated to be 0. Rice et al.0 Reference (17–22) Faroes Islands 917 Seychelles Average for two studies 711 Not applicable 15.0 Budtz-Jorgensen et al.056 mg/l. determined by dividing a maternal hair-mercury concentration of 14 mg/kg by the hair : blood ratio of 250.Table 10 Estimated maternal hair-mercury concentrations at no-observed-effect level (NOEL) and bench-mark-dose lower-confidence limit (BMDL) for neurotoxicity associated with exposure to methylmercury in utero Study N NOEL/BMDL (mg/kg maternal hair) 12. US National Research Council (2000). the Committee determined that a steady-state daily ingestion of methylmercury at 1. Despite an elimination half-life for methylmercury of approximately 2 months. the steady state concentration of mercury in blood can be related to average daily intake using a one-compartment model that incorporates refinements (20) to the original WHO (1990) formula (24). 2000.05) bw = body weight (65 kg for a pregnant female) d = dose (mg/kg of body weight per day) where The Committee used values appropriate to conversion during pregnancy. as follows: d= C¥b¥V A¥f¥bw C = mercury concentration in blood (mg/l) b = elimination rate constant (0. as the fetal period is considered to be the most vulnerable stage of life.

Potential human variability was taken into account by the application of adjust138 . Japan. This information included the results of studies performed in laboratory animals and humans.3–1. The Committee also evaluated information published between 1997 and 2003 on concentrations of mercury and methylmercury in various fish species. and use of biomarkers of exposure for methylmercury.2. fish is the only significant source of methylmercury in food.5 mg/kg of body weight per week for the five regional GEMS/Food diets and from 0. and life in utero the most sensitive period of exposure. and did not allow for interindividual variability in either the hair : blood ratio or in the elimination rate constant in the equation shown above.5 Estimated dietary intake At its fifty-third meeting.6 Evaluation The Committee evaluated new information which had become available since methylmercury was considered at the fifty-third meeting. The calculations made in the dose–response assessment are based on average values for each parameter. 7. as well as analyses of methylmercury intake by populations consuming large amounts of fish (>100 g per person per day). Older and larger predatory fish species and certain marine mammals contain the highest concentrations of methylmercury. New Zealand. concentrations of methylmercury are <0. the Committee updated its evaluations of national intakes. and epidemiological studies of the possible effects of prenatal exposure to methylmercury on child neurodevelopment. Generally. The Committee noted that overall methylmercury concentrations in fish species were similar to those considered at the fifty-third meeting and therefore concluded that the analyses of exposure conducted at the fifty-third meeting remained current. and Slovakia.have no appreciable adverse effects on offspring in these two study populations. and fish in particular. the Committee re-evaluated the safety of methylmercury-contaminated foods. These estimates range from 0. At its current meeting.2.1–2. but fish at the highest trophic levels may contain concentrations >5 mg/kg. Neurodevelopment was considered to be the most sensitive health outcome. The reevaluation included consideration of information on potential intake submitted by numerous national bodies. adding intake information submitted by Australia. For most populations.4 mg/kg.0 mg/kg of body weight per week for numerous national diets. 7. France.

and life in utero is the critical period for the occurrence of neurodevelopmental toxicity as a result of exposure to methylmercury.2) was applied to this figure to derive a PTWI of 1. including any analytical errors. the Committee concluded that the 139 .5) (23) to account for the total human interindividual variation for dose reconstruction (converting maternal blood concentration to a steady-state dietary intake).2(100. • Interindividual variation in pharmacokinetics should be taken into account when converting the steady-state concentration of mercury in maternal blood to an estimated daily intake. no uncertainty factor is needed to account for variation in vulnerability among subgroups. As the two study samples represent diverse populations. A total uncertainty factor of 6. As limited data were available which were specific to the study populations used in this assessment. although the ratios reported for humans in a limited number of studies were in the range of 137–585.5 (370/250). which is indicated by the differences in study means and by the limited individual data. • The available data on the hair : blood ratio show both interstudy and intersubject variability. This PTWI is considered sufficient to protect developing fetuses. the Committee recommended the use of a combined uncertainty factor of 3.3 (585/250). A steady-state intake of methylmercury of 1. while the ratio to the highest individual value (585) was 2.6 mg/kg of body weight.ment or uncertainty factors.4 (2 ¥ 3. and decided to apply a factor of 2 to the overall average of 250 to allow for the likely interindividual variation. Most of the published means are within a range of 140– 370. the most sensitive subgroup of the population.5 mg/kg of body weight per day was estimated to represent the exposure that would be expected to have no appreciable adverse effects on children. The ratio of the overall average (250) to the highest mean found (370) was 1. Pending reduction in the uncertainty associated with various aspects of the derivation of the steady-state intake from maternal hairmercury concentrations. In choosing the factors to apply to this intake estimate. Few data were available to the Committee on the range of individual hair : blood ratios. the Committee considered the following: • Neurodevelopment is a sensitive health outcome. No population-specific hair : blood ratios are available for the populations of the Faroe Islands or the Seychelles. The Committee concluded that the available data on the distribution of individual ratios were not adequate to allow derivation of a chemical-specific adjustment factor.

Since the PTWI for methylmercury was revised at the current meeting. to accommodate compounds with high intake evaluated by the “B-side” of the Procedure for the Safety Evaluation of Flavouring Agents. reference 100) be updated to reflect more modern technology. the Committee recommended that the PTWI for total mercury be revised. of which no more than 200 mg should be present as methylmercury. the Committee established a PTWI for total mercury of 300 mg/person. nutritious diet and that this should be appropriately considered in public health decisions to set limits for methylmercury concentrations in fish. This PTWI of 3. reference 30). head space chromatography and gas chromatography with capillary columns. • The Committee recommended that the PTWI for total mercury should be reviewed in light of the new PTWI established for methylmercury. Recommendations 1. • The Committee recommended a revision of the guidelines for the preparation of monographs for flavouring agents.3 mg/kg of body weight for methylmercury was confirmed at subsequent meetings. At its sixteenth meeting (Annex 1. The Committee considered whether a provisional tolerable monthly intake PTMI rather than a PTWI for methylmercury should be established. The Committee also reaffirmed its position that fish are an important part of a balanced. The Committee recognized that quillaia extracts differ from each other in their chemical composition and recommended that the Codex Committee on Food Additives and Contaminants consider 140 . 8. Future work • The Committee recommended that the General Method for the determination of residual solvents included in the Guide to Specifications (Annex 1. for example. but deferred its decision pending the outcome of the Joint FAO/WHO Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food. 9. • The Committee recommended that the evaluation of cadmium should be revisited after completion of the Project to Update the Principles and Methods for the Risk Assessment of Chemicals in Food.uncertainty factor could be refined and possibly reduced.

3. Mattock. such as the Codex Alimentarius Commission. At its fifty-ninth meeting. Scientific Committee on Food: Opinion of the Scientific Committee on Food on eucalyptol. 2000. Grande H. SCF/CS/FLAV/FLAVOUR/20 ADD2 Final. 1956 (WHO Technical Report Series. 107). 4. the Committee recognized the need for a working definition of the term “flavouring agent”. 23 April 2002. At the present meeting. The Committee noted that several substances assigned high priority by the Codex Committee on Food Additives and Contaminants for review by the Expert Committee have a carcinogenic potential. Joint FAO/WHO Conference on Food Additives. Vaccine. the Committee noted that a range of regulatory definitions exist and that such a definition would need to be elaborated in an international forum. References 1. St Jean d’Ardières. 141 . Kamstrup S. expressed on 17 April 2002. World Health Organization. Acknowledgements The Committee wishes to thank Dr H. France. 2. 3.adopting individual International Numbering System (INS) numbers for each type of extract.18: 2244–2249. 1956 (FAO Nutrition Meetings Report Series. for her assistance in the preparation of the report. Food and Agriculture Organization of the United Nations. the important role that the recommendations of the Committee play in the development of international food standards and of regulations in many countries. In view of the large number of food additives and contaminants requiring evaluation or re-evaluation. and the need for maintaining consistency and continuity within the Committee. Rome. 11). Doberti A. 2. San Martin R. it is strongly recommended that the meetings of the Joint FAO/WHO Expert Committee on Food Additives continue to be held at least once yearly to evaluate these substances. The Committee recommended that these substances should not be evaluated by JECFA before the International Programme on Chemical Safety (IPCS) Harmonization Project has reached a conclusion on how to assess the dose–response relationship for such substances. Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A. No. No. Dalsgaard K. Geneva.

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10. 8. WHO Food Additives Series. 153. 164. 2001. WHO Technical Report Series No. 2003. 2003. 160. 152. 909. 901. 166.149. 918. Compendium of food additive specifications: addendum 8. 41/14. 2001. Toxicological evaluation of certain veterinary drug residues in food. 157. No. 162. 163. WHO Food Additives Series. No. 9. 41/15. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). Compendium of food additive specifications: addendum 9. 2001. 913. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). 150. WHO Food Additives Series. FAO Food and Nutrition Paper No. No. No. FAO Food and Nutrition Paper. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series No. No. WHO Food Additives Series. WHO Technical Report Series. 2002. 155. 152 . 2002. Safety evaluation of certain food additives and contaminants. WHO Technical Report Series. No. 158. Add. 50. 2000. No. No. 52. FAO Food and Nutrition Paper. 151. 48. 2002. 154. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/ WHO Expert Committee on Food Additives). 2002. 2001. 46. No. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series. 2003. Evaluation of certain veterinary drug residues in food (Sixtieth report of the Joint FAO/WHO Expert Committee on Food Additives). 159. Evaluation of certain food additives and contaminants (Fifty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). 52. No. Residues of some veterinary drugs in animals and foods. Add. Add. 2002. 2002. 47/FAO Food and Nutrition Paper 74. 2002. FAO Food and Nutrition Paper. 911. Safety evaluation of certain food additives and contaminants. 906. No. No. 49. No. 51. FAO Food and Nutrition Paper. 52. 156. Compendium of food additive specifications: addendum 10. Safety evaluation of certain mycotoxins in food. 2003. Residues of some veterinary drugs in animals and foods. 2002. No. WHO Technical Report Series. WHO Food Additives Series. WHO Technical Report Series. Safety evaluation of certain food additives and contaminants. 165.

not acid-precipitated) — “Annatto G” Curcumin Diacetyltartaric and fatty acid esters of glycerol D-Tagatose Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae Mixed xylanase. T R.5% is is norbixin)c 0–0. T 0–7 (temporary). T 0–4 (temporary). for preparations containing not less than 85% pigment (as norbixin)c No ADI established. produced by a strain of Humicola insolens N R. since no data on toxicity were available R. licheniformis Annatto extract (solvent-extracted bixin) — “Annatto B”b Annatto extract (solvent-extracted norbixin) — “Annatto C” Annatto extract (oil-processed bixin suspension) — “Annatto D” Annatto extract (aqueousprocessed bixin) — “Annatto E” Annatto extract (alkaliprocessed norbixin) — “Annatto F” Annatto extract (alkaliprocessed norbixin. of which not more than 2. other toxicological information and information on specifications Food additives evaluated toxicologically Food additive Specificationsa Acceptable daily intake (ADI in mg/kg of body weight) and other toxicological recommendations ADI “not specified”d a-Amylase from Bacillus licheniformis containing a genetically engineered a-amylase gene from B.4 (temporary).Annex 2 Acceptable daily intakes. since no data on toxicity were available R — R N 0–3 0–50 0–125 (temporary) ADI “not specified”d N ADI “not specified”d 153 . T R. for preparations containing not less than 85% pigment (as bixin. of which not more than 7% is norbixin)c 0–0.4 (temporary). for a preparation containing not less than 35% pigment (as norbixin)c No ADI established. b-glucanase enzyme preparation. for a preparations containing not less than 25% pigment (as bixin. T R. T R.

in the opinion of the Committee. An additive meeting this criterion must be used within the bounds of good manufacturing practice. D. R: existing specifications revised. the establishment of an ADI expressed in numerical form is not deemed necessary. biochemical. It is not expressed in relation to content of bixin and/or norbixin. For that reason. on the basis of the available data (chemical. To ensure clarity of the text. represent a hazard to health.e. and for the reasons stated in the individual evaluations. quillaia extract (type 2): saponin content of 75–90%. does not. as employed in the submitted information. 154 . F. toxicological and other) and the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effects and from its acceptable background levels in food. Quillaia extract (type 1): saponin content of 20–26%. to refer to the different extracts under evaluation. E. it should not conceal food of inferior quality or adulterated food. and it should not create a nutritional imbalance. The ADI is established for the extract as tested biologically and specified. C. T: the existing specifications are tentative. T: tentative specifications.Food additives evaluated toxicologically Food additive Specificationsa Acceptable daily intake (ADI in mg/kg of body weight) and other toxicological recommendations 0–2 0–50 0–5 No ADI established due to limited information on the qualitative and quantitative composition ADI “not specified”d Neotame Polyvinyl alcohol Quillaia extract (type 1)e Quillaia extract (type 2)e N N R N Xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum a b N c d e N: new specifications prepared. Food additives considered for specifications only Food additive b-Carotene from Blakeslea trispora Magnesium silicate Monomagnesium phosphate Natamycin Sucrose esters of fatty acids Talc Trisodium diphosphate a Specificationsa R R S. it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect. ADI “not specified” is used to refer to a food substance of very low toxicity which. information required. the Committee adopted for this report the designations B. T R R R R. G. S: specifications maintained (revision considered but not required). i. T R: existing specifications revised.

4-Heptadienal (E. Alicyclic. B.Flavouring agents evaluated by the Procedure for the Safety Evaluation of Flavouring Agents A.E )-2.4-Octadien-1-ol trans.4-Pentadienal (E. 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 Specificationsa N N N N N N N N N N N N N N N Conclusion based on current intake No safety concern No safety concern No safety concern See footnoteb No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern 155 .trans-2. Aliphatic di.4-Nonadienal Nona-2-trans-6-cis-dienal 2-trans-6-trans-Nonadienal No. 1157 1158 1159 1160 1161 1162 1163 1164 Specificationsa N N N N N N N N Conclusion based on current intake No safety concern No safety concern No safety concern No No No No No safety safety safety safety safety concern concern concern concern concern 1165 1166 1167 1168 1169 1170 1171 1172 N N N N N N R N No safety concern No safety concern No safety concern No No No No No safety safety safety safety safety concern concern concern concern concern N: new specifications prepared. acids.6-Nonadien-1-ol 2.4-Nonadien-1-ol 2.4-Octadienal 2-trans.E )-2.6.trans-2.and trienals and related alcohols.4-Hexadienoic acid Methyl sorbate Ethyl sorbate 2. alicyclic-fused and aromatic-fused ring lactones Flavouring agent 4-Hydroxy-4-methyl-5-hexenoic acid g-lactone (+/-) 3-Methyl-g-decalactone 4-Hydroxy-4-methyl-7-cis-decenoic acid g-lactone Tuberose lactone Dihydromintlactone Mintlactone Dehydromenthofurolactone (+/-)-(2.6-trans-Octadienal 2.6-Trimethyl-2hydroxycyclohexylidene) acetic acid g-lactone Sclareolide Octahydrocoumarin 2-(4-Methyl-2-hydroxyphenyl)propionic acid g-lactone 3-Propylidenephthalide 3-n-Butylphthalide 3-Butylidenephthalide Dihydrocoumarin 6-Methylcoumarin a No. R: revised specifications.4-Hexadienal (E.E )-2. and esters Flavouring agent 2.4-Hexadien-1-ol trans.

1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 Specificationsa N N N N N N N.4-Dodecadienal 2-trans-6-cis-Dodecadienal 2-trans-4-cis-7-cis-Tridecatrienal a b No.Z )-2.7-decatrienoate Propyl 2. new specifications prepared.4-Dimethyl-2-pentenoic acid 2-Methylheptanoic acid Isobutyl angelate 2-Butyl-2-butenal 2-Isopropyl-5-methyl-2-hexenal 2-Ethyl-2-heptenal 2-Methyl-2-octenal 4-Ethyloctanoic acid dl-Citronellol Citronellal 3.4-Undecadienal trans. An ADI of 0–25 mg/kg of body weight was established at the seventeenth meeting. acids. Aliphatic branched-chain unsaturated alcohols.and trienals and related alcohols. and related esters Flavouring agent (+/-) 2-Methyl-1-butanol 3-Methyl-2-buten-1-ol 2-Methyl-2-butenal 3-Methyl-2-butenal Ammonium isovalerate 3-Methylcrotonic acid trans-2-Methyl-2-butenoic acid Isobutyl 2-butenoate 2-Methylallyl butyrate 4-Methyl-2-pentenal 2-Methyl-2-pentenal 2-Methyl-2-pentenoic acid 2.trans-2. Aliphatic di.B.4-Decadien-1-ol 2-trans.4. T N N N N N N N N N N N N N.6-Nonadien-1-ol acetate (E.7-Dimethyl-6-octenoic acid Rhodinol 156 No. aldehydes. T R N N N Conclusion based on current intake No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern See footnoteb No safety concern No safety concern No safety concern .E )-2. The ADI was maintained and the use of the chemical as a flavouring agent subsumed in the ADI.4-trans-Decadienal Methyl (E )-2-(Z )-4-decadienoate Ethyl trans-2-cis-4-decadienoate Ethyl 2. 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 Specificationsa N N N N N N N N N N N Conclusion based on current intake No No No No No No No No No No No safety safety safety safety safety safety safety safety safety safety safety concern concern concern concern concern concern concern concern concern concern concern N. acids. and esters Flavouring agent (E. R: existing specifications revised. C.4-decadienoate 2.

2-Dimethoxybenzene m-Dimethoxybenzene p-Dimethoxybenzene 3.0. Aliphatic branched-chain unsaturated alcohols. The ADI was maintained and the use of the chemical as a flavouring agent subsumed in the ADI.3.4-Dimethoxy-1-vinylbenzene Benzyl ethyl ether Benzyl butyl ether No safety concern No safety concern No safety concern No No No No No No No No No No No No No safety safety safety safety safety safety safety safety safety safety safety safety safety concern concern concern concern concern concern concern concern concern concern concern concern concern 157 .9))tridecane Anisole o-Methylanisole p-Methylanisole p-Propylanisole 2.7. A group ADI of 0–0. D.10-dodecatrienal 12-Methyltridecanal Farnesol a No.0(4. T: the existing. citronellol.6.6-Trimethyl-6-vinyltetrahydropyran Tetrahydro-4-methyl-2(2-methylpropen-1-yl)pyran Theaspirane Cycloionone 1.11-Trimethyl-2. 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 Specificationsa N N N N N N N N N N N N N N N N N N N N N N R Conclusion based on current intake No No No No No No No safety safety safety safety safety safety safety concern concern concern concern concern concern concern sec-Butyl ethyl ether 1-Ethoxy-3-methyl-2-butene 1.5.2.C. 1223 1224 1225 1226 1227 1228 1229 1230 Specificationsa N N R N N N N N Conclusion based on current intake No safety concern No safety concern See footnoteb No safety concern No safety concern No safety concern No safety concern No safety concern b N: new specifications prepared.11-dodecatrienal 3. geranyl acetate. R: existing specifications revised.6-Dimethyl-10-methylene2. aldehydes. linalool and linalyl acetate at the twenty-third meeting. was established for citral. acids.5.5 mg/kg of body weight expressed as citral. Aliphatic and aromatic ethers Flavouring agent No. and related esters Flavouring agent Geraniol Nerol Citral 8-Ocimenyl acetate 2.9-Tetramethyl-13-oxatricyclo (8.4-Dimethylanisole 1-Methyl-3-methoxy-4isopropylbenzene Carvacryl ethyl ether 1.4-Cineole Eucalyptol Nerol oxide 2. new or revised specifications are tentative and new information is required.6.

aldehydes. acids and related esters Flavouring agent Isoprenyl acetate 4-Pentenyl acetate 3-Hexenal 3-Hexenyl formate Ethyl 5-hexenoate cis-3-Hexenyl propionate cis-3-Hexenyl isobutyrate (Z)-3-Hexenyl (E)-2-butenoate cis-3-Hexenyl tiglate cis-3-Hexenyl valerate 3-Hexenyl 2-hexenoate (Z)-4-Hepten-1-ol Ethyl cis-4-heptenoate (Z)-5-Octenyl propionate 158 No. new specifications prepared.D.T N N N N N N N N N Conclusion based on current intake No No No No No No No No No No No No No No safety safety safety safety safety safety safety safety safety safety safety safety safety safety concern concern concern concern concern concern concern concern concern concern concern concern concern concern . Hydroxypropenylbenzenes Flavouring agent Isoeugenol Isoeugenyl formate Isoeugenyl acetate Isoeugenyl phenylacetate Propenylguaethol 4-Propenyl-2. R. existing specifications revised E. F. new specifications prepared. the existing. Aliphatic and aromatic ethers Flavouring agent Methyl phenethyl ether Diphenyl ether Dibenzyl ether b-Naphthyl methyl ether b-Naphthyl ethyl ether b-Naphthyl isobutyl ether a No. new or revised specifications are tentative and new information is required. 1254 1255 1256 1257 1258 1259 Specificationsa N N R N N N Conclusion based on current intake No No No No No No safety safety safety safety safety safety concern concern concern concern concern concern N. T. 1260 1261 1262 1263 1264 1265 1266 1267 1268 Specificationsa N N N N. unconjugated alcohols. Linear and branched-chain aliphatic unsaturated.6-dimethoxyphenol Isoeugenyl methyl ether Isoeugenyl ethyl ether Isoeugenyl benzyl ether a No. T. T N N N N N Conclusion based on current intake No No No No No No No No No safety safety safety safety safety safety safety safety safety concern concern concern concern concern concern concern concern concern N. 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 Specificationsa N N N N N.

Linear and branched-chain aliphatic unsaturated.6-Nonadien-1-ol (E)-3. the existing. Flavouring agents considered for specifications only No. T Conclusion based on current intake No safety concern No safety concern No safety concern No No No No No safety safety safety safety safety concern concern concern concern concern erythro and threo-3-Mercapto-2methylbutan-1-ol (+/-)2-Mercapto-2-methylpentan-1-ol 3-Mercapto-2-methylpentan-1-ol (racemic) 3-Mercapto-2-methylpentanal 4-Mercapto-4-methyl-2-pentanone (+/-) Ethyl 3-mercaptobutyrate Ethyl 4-(acetylthio)butyrate spiro(2. G. the existing.F. new or revised specifications are tentative and new information is required.5-Trithiahexane Diisopropyl trisulfide a 1297 1298 1299 1300 N N N N No No No No safety safety safety safety concern concern concern concern N.T R R R 159 .3¢(1¢-oxa-2¢-methyl)-cyclopentane) 2-(Methylthio)ethanol Ethyl 5-(methylthio)valerate 2. acids and related esters Flavouring agent (Z.4-Dithia-1-methyl-8oxabicyclo(3. new specifications prepared. new or revised specifications are tentative and new information is required.3. aldehydes.(Z)-6-Nonadien-1-ol (E. unconjugated alcohols.6-Nonadien-1-ol acetate 9-Decenal 4-Decenoic acid cis-4-Decenyl acetate a No.3. T. new specifications prepared.Z)-3.T R R. 1283 1284 1285 1286 1287 1288 Specificationsa N N N N N N Conclusion based on current intake No No No No No No safety safety safety safety safety safety concern concern concern concern concern concern N. 42 53 54 55 56 57 60 Flavouring agent Isoamyl formate Citronellyl formate Geranyl formate Neryl formate Rhodinyl formate Citronellyl acetate Rhodinyl acetate Specificationsa R R. Simple aliphatic and aromatic sulfides and thiols Flavouring agent No. T N N N N N.Z)-3. T.0)octane-3. 1289 1290 1291 1292 1293 1294 1295 1296 Specificationsa N N N.

3-Undecadione Ethylcyclo-pentenolone 3-Ethyl-2-hydroxy-4-methylcyclopent-2-en-1-one Specificationsa R R R R R.7-Dimethyl-1-octanol 2.T R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R.6-Dimethyl-4-heptanone 2.Flavouring agents considered for specifications only No.6-Dimethyl-6-hepten-1-ol 2.7-dimethyloctanoic acid lactone 3-Heptyldihydro-5-methyl-2(3H)-furanone 3.6-Dimethyl-4-heptanol cis-5-Octen-1-ol cis-5-Octenal cis-6-Nonenal 9-Undecenal Linoleic and linolenic acid (mixture) Methyl cis-4-octenoate Ethyl cis-4-octenoate Methyl linoleate & Methyl linolenate (mixture) 2.3-Pentadione 2.T R R R R R . 61 62 65 66 68 71 73 95 98 101 104 107 110 112 117 119 124 170 180 205 212 237 244 272 302 303 322 323 325 329 332 337 338 346 348 349 358 360 384 385 396 397 399 409 410 417 419 422 160 Flavouring agent Citronellyl propionate Geranyl propionate Citronellyl butyrate Geranyl butyrate Rhodinyl butyrate Citronellyl isobutyrate Neryl isobutyrate Heptanal Octanal Nonanal Decanal Undecanal Lauric aldehyde Myristaldehyde Propyl formate n-Amyl formate Isobutyl formate n-Amyl heptanoate Methyl laurate Methyl 2-methylbutyrate 2-Methylbutyl 2-methylbutyrate 6-Hydroxy-3.6-Dimethyl-5-heptenal Linalyl formate Linalyl propionate b-Damascone a-Damascone Dehydrodihydroionone Dehydrodihydroionol Methyl-b-ionone 3-Hydroxy-2-pentanone 2.

3.2 632.T R R R R R R R.2 633. sodium salt 3-Methyl-2-oxopentanoic acid. sodium salt Linalyl cinnamate Terpinyl cinnamate p-Tolyl laurate 2-(Methylthio)methyl-2-butenal 2.T R R R R R R R R.T R R R R R.8-Dithianon-4-ene-4-carboxaldehyde Methylthiomethyl butyrate Methylthiomethyl hexanoate Ethyl 3-(methylthio)butyrate S-Methyl 4-methylpentanethioate S-Methyl hexanethioate 1-Methylthio-2-propanone Di(butan-3-one-1-yl) sulfide S-Methyl benzothioate 2-Ethylhexanethiol 4-Methoxy-2-methyl-2-butanethiol 3-Mercaptohexyl hexanoate 1-Mercapto-2-propanone 2-Keto-4-butanethiol Allyl methyl disulfide Methyl 1-propenyl disulfide Propenyl propyl disulfide Methyl 3-methyl-1-butenyl disulfide Methyl ethyl trisulfide Allyl methyl trisulfide Methyl 2-hydroxy-4-methylpentanoate Citronelloxyacetaldehyde Ethyl 2.T R R R R.T R R R R R R R R.T R R.2 668 669 704 470 471 473 479 480 488 489 495 502 504 519 548 556 557 559 568 569 570 571 583 586 590 592 603 604 605 615 625 628 735 737 918 923 924 937 a Flavouring agent 5-Ethyl-2-hydroxy-3-methylcyclopent-2-en-1-one Piperitone l-Menthol ethylene glycol carbonate 2-Methylthioacetaldehyde 4-(Methylthio)butanal 3-Methyl-2-oxobutanoic acid. sodium salt 4-Methyl-2-oxopentanoic acid.T R R R R. 423 435 443 465 468 631.4-dioxohexanoate 3-(Hydroxymethyl)-2-heptanone 1. existing specifications revised. or revised specifications are tentative and new information is required.T R.T R. existing specifications were maintained.6-Trimethylphenol Glyceryl monostearate Glycerol 5-hydroxydecanoate Glycerol 5-hydroxydodecanoate Pyruvaldehyde Specificationsa R R R R R R.3-Nonanediol acetate (mixed esters) Butyl ethyl malonate Dibutyl sebacate Ethyl aconitate (mixed esters) 2-Phenylphenol 2. new. T. S. 161 .T R. the existing.Flavouring agents considered for specifications only No.

Evaluation of a water-treatment agent Agent Sodium dichloroisocyanurate (NaDCC) Specificationsa N Tolerable daily intake (TDI) and other toxicological recommendations 0–2.6 mg/kg of body weight 162 . new specifications prepared Contaminants Contaminant Cadmium Methylmercury Tolerable intake and other toxicological recommendations Provisional tolerable weekly intake (PTWI) of 7 mg/kg of body weight (maintained) Provisional tolerable weekly intake (PTWI) of 1. applicable for intake from drinking-water treated with NaDCC for the purpose of disinfection a N. new specifications prepared Evaluation of a nutritional source of iron Source Ferrous glycinate (processed with citric acid) Specificationsa N Toxicological recommendation Suitable for use as a source of iron for supplementation and fortification. providing that the total intake of iron does not exceed the provisional maximum tolerable daily intake of 0.8 mg/kg of body weight per day a N.0 mg/kg of body weight for anhydrous NaDCC.

trisodium diphosphate Information on the method for loss on drying for the hydrates is necessary in order to express the assay on the dry basis for the above additives.Annex 3 Further information required or desired Annatto extracts The Committee requested additional information to clarify the role that non-pigment components of the extract play in the expression of the qualitative and quantitative differences in toxicity of the various extracts. by 2006. D-Tagatose The Committee requested information on the histological examination of the adrenals. In addition. Monomagnesium phosphate. Requested by end of 2004. 163 . that contains norbixin. such as Annatto F. the Committee requested data on the reproductive toxicity of an extract. kidneys and testes of the rats from the 2-year study.

1983). alicyclic-fused and aromatic-fused ring lactones 1158 (+/-) 3-Methyl-..4hexandione (No. 3.6. ..dodecalactone (No. dihydro-5-(2-octenyl)-(Z) 1160 Tuberose lactone 1164 90% (+/-)-(2. 22–30% 2(3H)-Furanone. 2.8decanedione...-lactone 3. 249) and concluded they were of no safety concern at current levels of intake. in a 90-day study in rats (Gaunt et al.6. The Committee has evaluated . 235) and 2(3H)furanone..9-Dimethyl-3. 1965).5–4. Alicyclic. 3.5% 2.5% 4-Hydroxy-5..-ionone (No.8-decadione has not been evaluated by the Committee However.6-oxo . Flavouring agent A.-Trimethyl-2hydroxycyclohexylidene)acetic acid . A NOEL of 10 mg/kg bw per day was reported for the structurally related substance.9-Dimethyl 3.. 389). dihydro-5-(2-octenyl)-(Z) (No.-decalactone 94% 1–2% heptan-1-ol (sum of cis and trans isomers) 45% 28–35% . 94) and concluded it was of no safety concern at current levels of intake. respectively (Oser et al. 1969).5–4. Another 90-day study reported NOELs of 11 and 13 mg/kg bw per day for males and females. . 413) was >17 mg/kg bw per day in a 90-day study in rats (Posternak et al.-ionone The Committee has evaluated heptan-1-ol (No.-Dodecalactone. the NOEL for another diketone.164 Annex 4 Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of 95% or less Minimum assay value (%) Secondary components Comments on secondary components No.

respectively (NTP. alicyclic. A 98-day study for the structurally related material 2.4hexadienoic showed a NOEL of 15 and 60 mg/kg bw for male and female rats. 1972).4-Heptadienal 92% 2–4% (E. di. The (E. respectively (NTP.E)-2.Z)-2. 1180 (E. 1973) and ALDH (Feldman and Weiner. 2001b). 1972) and entry in to fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (Nelson and Cox..4-heptadienoic expected to share the same metabolic acid fate as the primary material.E)-2. linear •. 2001a). .Z) isomer is expected to share the same metabolic fate as the (E. Aliphatic. Both secondary components are 2–4% 2.4-Heptadienoic acid is a substrate of the fatty acid cycle and is metabolized and excreted primarily as carbon dioxide and water (Nelson and Cox..4-Octadien-1-ol 94% 2–4% (E.4-isomer. 2000). 2. A 90 day study for the structurally related material 2.and trienals and related alcohols.E) form by the action of 3hydroxy acyl CoA epimerase and oxidized to 2.4hexadienal showed a NOEL of 15 and 60 mg/kg bw per day for male and female rats.165 B.Z) isomer is expected to be converted to the (E. acids and esters 1179 (E.4-heptadienoic acid by aldehyd dehydrogenase (ALDH) (Feldman and Weiner. 2000).4-isomer The (E.E) isomer: conversion to the corresponding carboxylic acid by alcohol dehydrogenase (ADH) (Pietruzko et al.-unsaturated.Z)-2.

4-Decadien-1-ol 3–5% (E.Z) isomer is expected to share the same metabolic fate as the (E. see No.E)-2.Z) isomer A-90 day study for the structurally related material (E. It is expected to be oxidized and completely metabolized in the fatty acid cycle (see No. Minimum assay value (%) Secondary components Comments on secondary components Flavouring agent 1183 92% 4–5% 2-nonen-1-ol 2.4-Nonadien-1-ol (No. 1997). The alcohol is converted to the corresponding carboxylic acid by ADH and ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (see Nos 1179 and 1180 above). 1–2% 2-nonen-1-ol 2. 1179 and 1180 above).4-Nonadienal 1189 92% (E.E) isomer.166 No. 2.E)-2. . 2-Nonen-1-ol is scheduled to be evaluated by the Committee in 2004. 1183 above. 2-Nonen-1-ol.4-decadienal established a NOEL of 100 mg/kg bw per day (NTP. The (E.4-nonadien-1-ol. It is expected to be oxidized to the corresponding acid and metabolized in the fatty acid cycle and excreted primarily as carbon dioxide and water (see Nos. 1183) has been evaluated by the Committee.4-Nonadien-1-ol 1185 89% 5–6% 2. 1179 and 1180 above).

E) and (E. A NOEL of 167 . 3–4% acetone plus trace of isopropanol.9 mg/kg bw per day (Damske et al..E) isomer The (Z. Readily hydrolysed to methanol and (E.Z). 1980). Methanol is oxidized in vivo to formic acid. The NOEL for (E. 1980) in separate 90-day studies.5% unknown . cis trans.4-decadienal was 100 mg/kg bw per day (NTP.Z) isomers are expected to share the same metabolic fate as the (E. 1997) and 33. 1191 93% Methyl (E)-2-(Z)-4-decadienoate 5–7% (E..E)-2.E) isomer. 1180 above). 1997) and 33. (Z.E)-2. Acetone (No. 0. Separate 90-day studies on the related substance (E.4-decadienoic acid which is a substrate for the fatty acid cycle (See No. Both substances were concluded to be of no safety concern at current intake levels.4-trans-Decadienal 3–4% mixture of cis cis. 277) have been evaluated by the Committee.4decadienals.1190 89% 2-trans. 139) and isopropanol (No.9 mg/kg bw per day (Damske et al. The Committee has evaluated formic acid (No. The aldehyde is converted to the corresponding carboxylic acid by ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (See Nos 1179 and 1180 above). 79) and concluded that it was of no safety concern at current intake levels.4-decadienal established NOELs of 100 mg/kg bw per day (NTP. and trans cis 2.E)-2.

9 mg/kg bw per day (Damske et al. In a 63-day study in rats.4-decadienal established NOELs of 100 mg/kg bw per day (NTP. trans-4-decadienoate 1196 85% trans. Minimum assay value (%) Secondary components Comments on secondary components Flavouring agent 1192 90% Ethyl trans-2-cis-4-decadienoate 5–10% ethyl trans-2. 1980).E)-2. 1969). Readily hydrolysed to ethanol and (E.168 No..E) isomer. Ethanol is oxidized in vivo to acetic acid. 1997) and 33. The alcohol is converted to the corresponding carboxylic acid by ADH and ALDH and then enters the fatty acid cycle where it is metabolized and excreted primarily as carbon dioxide and water (see Nos 1179 and 1180 above). 81) and concluded that it was of no safety concern at present intake levels.trans-2.4-Dodecadienal 11–12% 2-trans-4-cis isomer >400 mg/kg bw per day for formic acid was established in a 2-year rat study (Malorny. . The (E. The Committee has evaluated acetic acid (No. the NOEL for acetic acid was 350 mg/kg bw per day (Pardoe.Z) isomer is expected to share the same metabolic fate as the (E. Separate 90-day studies on the related substance (E. 1180 above). 1952).4-decadienoic acid which is a substrate for the fatty acid cycle (see No.E)2.

5–4. 83) and propionic 3.5–2.4-decadienal were 100 mg/kg bw per day (NTP. 1997) and 33.4-decadienal were 100 mg/kg bw per day (NTP. 259) (Amoore.E)2. 1978). 6% 3-cis-7-cistridecadienol. 169 . All secondary materials are expected to be oxidized to the corresponding acids and enter the fatty acid cycle where they will be metabolized and excreted primarily as carbon dioxide and water (See Nos 1179 and 1180 above).4-Dimethyl-2-pentenoic acid 92% 5–7% 4-methyl-24-Methyl-2-methylenevaleric acid has not (sum of isomers) methylenevaleric acid been evaluated previously.9 mg/kg bw per day (Damske et al. 1997) and 33. Aliphatic branched-chain. A 90-day study of oral administration in rats established a NOEL of >2500 mg/kg bw per day for the structurally related material. C. 5% 2-trans7-cis-tridecadienal.5% propionic acid acid (No.9 mg/kg bw per day (Damske et al. Propionaldehyde (No. 1211 2. acids. aldehydes. and related esters 1209 2-Methyl-2-pentenal 92% 1. saturated and unsaturated alcohols. 84) have been evaluated by the Committee. It was concluded that both substances were of no safety concern at current intake levels. isovaleric acid (No..5% propionaldehyde. NOELS for the related substance (E. 3% 2-trans-4-trans-7-cistridecatrienal NOELS for the related substance (E.. 1980) in separate 90-day studies. 1980) in separate 90-day studies.1198 71% 2-trans-4-cis-7-cis-Tridecatrienal 14% 4-cis-7-cis-tridecadienol.E)2.

1% citronellal 90% (of total alcohols as C10H20O) Flavouring agent 1219 dl-Citronellol 1220 Citronellal 85% of aldehydes 12–14% mixture of terpenoid as C10H18O materials: mainly 1. 2001b). 2-Isopropylidene-5methylcyclohexanol. In a 28-day rat study.170 No. In a 2-year study. 1987b). a terpene alcohol.200 mg/kg bw per day for males and females. 1967). 1. a structurally related material to citronellal. 1% citronellyl acetate. Roe et al.and 28-week studies in rats. citronellyl acetate and other naturally occurring terpenes Geraniol. Minimum assay value (%) Secondary components Comments on secondary components 5–8% di-unsaturated and saturated C10 terpene alcohols. exhibited a NOEL of 100 mg/kg bw per day in male and female rats (NTP. No. No.. respectively (NTP. In a 14-day oral rat study isopulegol was reported to . This corresponds to an estimated daily dose of 580 mg/kg bw for citronellyl acetate. respectively (Hagan et al. 1. exhibited NOELs of >1000 and >100 mg/kg bw per day in 16. 755) and concluded that it was not of safety concern at current intake levels. A NOEL of 2000 mg/kg bw per day was reported when rats were fed a mixture of 71% geranyl acetate and 29% citronellyl acetate for two years (NTP.8-Cineole (eucalyptol. In an 80week mouse study a NOEL of 32 mg/kg bw per day was reported (1979). linalool. 1234) has been evaluated by the Committee.8-cineole exhibited NOELs of 300 and 1. 1987a). citral.8-cineole.. The Committee has evaluated 2isopropylidene-5-methylcyclohexanol (isopulegol.

356) has been evaluated by the Committee. 58) and 29% citronellyl acetate (No. citronellyl. exhibited a NOEL of 100 mg/kg bw per day in male and female rats (NTP. linalool exhibited a NOEL of >50 mg/kg bw per day (Oser. Citronellal (No. 171 . being the cis isomer of geranyl acetate is expected to follow similar metabolic pathways and exhibit similar toxicologic potential. This corresponds to an estimated daily dose of 580 mg/kg bw for citronellyl acetate. In a 2-year study. and geraniol. 57) for 2 years (NTP. 1985). Neryl acetate. nerol. and geranyl acetate esters and other naturally occurring terpenes 3. See No.1221 90% 5–8% citronellal. 2001b). 1220 above for geranyl and citronellyl acetate.1967).7-Dimethyl-6-octenoic acid have a NOEL of 250 mg/kg bw per day (Imaizumi et al. It was concluded that linalool was not a safety concern at current intake levels. Linalool (No. In an 84-day study in rats. neryl. The naturally occurring terpenoid esters are expected to hydrolyse in vitro to the acetic acid and the corresponding terpene alcohols citronellol. 1220) has been evaluated by the Committee. 1987b).. A NOEL of 2000 mg/kg bw per day was reported when rats were fed a mixture of 71% geranyl acetate (No. the structurally related material citral.

and geranyl acetate esters and other naturally occurring terpenes D.No. and geranyl acetate esters and other naturally occurring terpenes 8–10% terpene esters: mainly citronellyl. Minimum assay value (%) Secondary components Comments on secondary components See No. 1221 above. which concluded that it was not a safety concern at current intake levels.8-cineole 1253 93% Benzyl butyl ether 2–5% benzyl alcohol 1.8-cineole exhibited NOELs of 300 and 1200 mg/kg bw per day for males and females. 1221 above. A 13week and a 2-year study in rats established NOELs of 100 and >200 mg benzyl alcohol/kg bw per day. 1989). 1979). neryl. Benzyl alcohol (No. 1987a)..4-Cineole 75% 20–25% 1. respectively (NTP.8-Cineole (eucalyptol. 1234) has been evaluated by the Committee. No. . Geraniol 15–17% terpenoid esters: mainly citronellyl. 82% (of total alcohols as C10H20O) Flavouring agent 172 1222 Rhodinol 1223 88% (of total alcohols as C10H18O) See No. 25) has been evaluated by the Committee. Aliphatic and aromatic ethers 1233 1. neryl. respectively (NTP. In a 28-day rat study 1. In an 80week mouse study a NOEL of 32 mg/kg bw per day was reported (Roe et al.

315) and 3-hexenoic acid (No. 1950). unsaturated. 0–1%. the resulting alcohol is oxidized to the corresponding acid that participates in normal fatty acid metabolism. 2. has been evaluated by the Committee. Linear and branched-chain aliphatic. in a 6-month study in rats (Tislow et al. Regardless of the position of unsaturation. hexenoate. . 317) and concluded that they are of no safety concern at current intake levels. respectively 3-Hexenyl-3-hexenoate and its isomers are expected to hydrolyse in vivo to mono unsaturated hexenol and mono unsaturated hexenoic acid. 336) hexenoate. unconjugated alcohols. 1969). 331). and 3 of hexenyl safety concern at current intake levels. aldehydes. 1279 3-Hexenyl 2-hexenoate 86% 6–8% 3-Hexenyl-3cis-3-Hexenyl-cis-3-hexenoate (No.173 F. acids and related esters 1271 3-Hexenal 80% 18–20% trans-2-Hexenal trans-2-Hexenal is scheduled to be (total of cis and evaluated by the Committee in 2004. and 0–0. A NOEL of 120–150 mg/kg bw per day was reported for cis-3-hexen-1-ol in a 98-day study of oral administration (Gaunt et al. The Committee has evaluated 3-hexen1-ol (No. 4–6%.. A NOEL of >400 mg/kg bw per day was reported for 10-undecenoic acid (No.. 1971).5% of isomers which concluded that it was not a 1. A trans isomers) 13-week study of oral administration in rats established a NOEL of 30 mg/kg bw per day for this material (Gaunt. a material that is structurally related to 3-hexenoic acid.

A 14day study in rats established a NOEL of >10 mg/kg bw per day for the structurally related substance 5-methyl5-hexen-2-one (No. the E isomer is expected to hydrolyse in vivo to 5octenol and propionic acid. 1987). *material was administered as part of a mixture.6-dodecadienal (No. 0. G. The Committee has evaluated propionic acid (No. Minimum assay value (%) Secondary components Comments on secondary components 2–3% (E)-5-Octenyl propionate.E) isomer Like the Z isomer. 322) and concluded that it was of no safety concern at current intake levels. 1131) and concluded that it posed no safety concern at current intake levels. 84) and concluded that it was of no safety concern at current intake levels. Simple aliphatic and aromatic sulfides and thiols 1293 4-Mercapto-4-methyl-248–50% pentanone 48–50% 4-methyl-3-penten-2one The Committee has evaluated 4-methyl-3penten-2-one (No. 1119) (Gill & van Miller. .Z)-2.174 No. 1197) (Edwards.5–1% (Z)-5Octenol 93% Flavouring agent 1282 (Z)-5-Octenyl propionate 1284 92% (E)-3. 1973).06 mg/kg bw per day was reported for the structurally related material (E. In a 28-day study in rats.(Z)-6-Nonadien-1-ol 6% (E. The Committee has evaluated cis-5octen-1-ol (No. a NOEL* of 2.

Hansen WH. Gangolli SD (1969) Acute (rat and mouse) and short-term (rat) toxicity studies on cis-3-hexen-1-ol.6-dodecadienal and 2. II. Gaunt IF. Gaunt IF. Edwards KB (1973) Biological evaluation of 2. Food and Cosmetics Toxicology. Food and Cosmetics Toxicology. 89-2599. Private Communication to the Flavor and Extract Manufacturers Association of the United States. Washington DC. Damske DR. 7: 451–459. National Toxicology Program (1989) Toxicology and carcinogenesis studies of benzyl alcohol in F344/N rats and B6C3F1 mice (gavage studies).8-cineole in Fischer 344 rats. 15-NTP Expt. Ford GP (1983) The short-term (90 day) toxicity of alphaand beta-ionones in rats.7tridecatrienal. Submitted to WHO by the Flavor and Extract Manufacturers Association of the United States. Beliles RP. Jones WI. Colley J. No. Lansdown ABG. Washington DC. van Miller JP (1987) Fourteen-day dietary minimum toxicity screen (MTS) in albino rats. Creasey M.4-decadienal administered by gavage to F344/N rats and B6C3F1 175 . 9: 332–339. 5: 141–157. US Government Printing Office. Nos 5014-02 and 5014-06. Fitzhugh OG. National Toxicology Program (1997) Final report on subchronic toxicity studies of 2. Unpublished report to the International Organisation of the Flavor Industry (IOFI). Mawartari K. 3: 307–317. Subacute and chronic toxicity. Mecler FJ. Grasso P. Colley J. Grasso P. Z Ernaehrungswiss. NTP Chem. Sugano M (1985) Effect of essential oils on the concentration of serum lipids and apolipoproteins in rats. NTP-TR343. 49: 2795–2796. Feldman RI. 2. NCTR Expt. Malorny G (1969) Acute and chronic toxicity of formic acid and formates. NTP-TR-252. NIH Publication No. National Toxicology Program (1987a) Twenty-eight day gavage and encapsulated feed study on 1. Chemical Senses and Flavour. Gould DH (1978) Synthetic flavors: efficiency and safety factors for sweaty and fishy odorants.4-Decadienal. Gill MW. Jenner PM. Journal of Agricultural and Biological Chemistry.4. Booth AN. 88-2508. Unpublished report. Gangolli SD (1971) Acute and short-term toxicity studies on trans-2-hexenal. Journal of Biological Chemistry. National Toxicology Program (1987b) Carcinogenesis studies of food grade geranyl acetate (71%) and citronellyl acetate (29%). Hagan EC. Imaizumi K. Gaunt IF. Performed by the British Industrial Biological Research Association (BIBRA). Wright M. NIH Publication No. Taylor JM (1967) Food flavourings and compounds of related structure. Hanada K. Private communication to the Flavor and Extract Manufacturers Association of the United States. 9: 775–786. (1972) Horse liver aldehyde dehydrogenase. Nos 380 and 439. Food and Cosmetics Toxicology. US Government Printing Office. Butler WH. Liverman JL (1980) 90-Day toxicity study in rats. 247(1): 260–266. Weiner H. Gumbmann MR. 4-week feeding study in rats.References for Annex 4 Amoore JE.

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